( H ) of surface of α - Actinin - positive NRVMs . A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group used for statistical . Two - way ANOVA followed by Bonferoni post - was used for statistical

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of the surface area of α-Actinin-positive NRVMs. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( I ) qRT - PCR of Acta1 Nppa in NRVMs . n = 4 : 4 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - was used for statistical

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4:4. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( A ) ChIP showing enrichment of NCoR1 in MEF2 binding regions on promoters of Acta1 Nppa in ventricular samples .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

(A) ChIP analysis showing enrichment of NCoR1 in MEF2 binding regions on the promoters of Acta1 and Nppa in ventricular samples.


( B ) ChIP of enrichment of MEF2a on promoters of Acta1 Nppa in ventricular samples . n = 3 : 3 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(B) ChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples. n=3:3.


ChIP of enrichment of HDAC4 ( C ) on promoters of Acta1 Nppa in ventricular samples . n = 4 : 4 for ( C )

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

ChIP analysis of enrichment of HDAC4 (C) on promoters of Acta1 and Nppa in ventricular samples. n=4:4 for (C)


ChIP of enrichment of HDAC5 ( D ) on promoters of Acta1 Nppa in ventricular samples . n = 3 : 3 for (

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

ChIP analysis of enrichment of HDAC5 (D) on promoters of Acta1 and Nppa in ventricular samples. n=3:3 for (D


ChIP of enrichment of , acetylated - Histone 4 ( ac - H4 ) ( E ) on promoters of Acta1 Nppa in ventricular samples . , n = 3 :

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

ChIP analysis of enrichment of , acetylated-Histone 4 (ac-H4) (E) on promoters of Acta1 and Nppa in ventricular samples. , n=3:3


ChIP of enrichment of ac - H3 ( F ) on promoters of Acta1 Nppa in ventricular samples . n = 3 :

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

ChIP analysis of enrichment of ac-H3 (F) on promoters of Acta1 and Nppa in ventricular samples. n=3:3


( G ) ChIP of Polymerase II enrichment on transcriptional regions of Acta1 Nppa in ventricular samples . n = 3 : 3 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(G) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples. n=3:3.


( A ) Western blotting of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1 - RIDs ( 1939 - 2453 ) - Flag .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(A) Western blotting analysis of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1-RIDs (1939-2453)-Flag.


( B ) VW / BW of mice 2 after sham operation or AAC . n = 7 : 7 : 12 : 12 .

O O O O O O O O O O O O O O O O O O O O O O O O O

(B) VW/BW of mice 2 weeks after sham operation or AAC. n=7:7:12:12.


( C ) Representative H & E of cross sections of left ventricles . Scale bar : 10μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 10μm.


( D ) Representative WGA . Scale bar : 10μm .

(D) Representative WGA staining. Scale bar: 10μm.


( E ) of cardiomyocyte . n = 6 : 6 : 7 : 8 .

(E) Quantification of cardiomyocyte size. n=6:6:7:8.


( F ) qRT - PCR of hypertrophy - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:8:11:11.


( G ) Representative picrosirius red of cross sections of left ventricles . Scale bar : 20μm .

(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 20μm.


( H ) of fibrotic areas . n = 5 : 5 : 7 : 7 .

(H) Quantification of fibrotic areas. n=5:5:7:7.


( I ) qRT - PCR of fibrosis - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

(I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:8:11:11.


( J ) Ejection fraction measured before , 2 4 after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - was used for statistical .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Ejection fraction measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( K ) Fractional shortening measured before , 2 4 after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - was used for statistical .

(K) Fractional shortening measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


A TUNEL comparing apoptotic in Miwi + / - Miwi - / - testis during diakinesis . Blue , DAPI ; Green , TUNEL labeling . Asterisk indicates TUNEL - positive stained in meiotic metaphase stage . Scale bars , 10 μm .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A TUNEL assay comparing apoptotic cells in Miwi+/- and Miwi-/- testis during diakinesis. Blue, DAPI; Green, TUNEL labeling. Asterisk indicates TUNEL-positive stained cells in the meiotic metaphase stage. Scale bars, 10 μm.


B , C Meiotic metaphase I plate in FACS - sorted Miwi heterozygous ( B ) knockout ( C ) spermatocytes . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bars , 10 μm . D Frequency of abnormal arrangement in meiotic metaphase I plate . , Miwi heterozygous ; gray , Miwi knockout . Three mice were used for each genotype , total numbers of counted metaphase I plate in Miwi heterozygous knockout are 113 181 , respectively .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm. D Frequency of abnormal arrangement in the meiotic metaphase I plate. White, Miwi heterozygous; Light gray, Miwi knockout. Three mice were used for each genotype, total numbers of counted metaphase I plate in Miwi heterozygous and knockout are 113 and 181, respectively.


B , C Meiotic metaphase I plate in FACS - sorted Miwi heterozygous ( B ) knockout ( C ) spermatocytes . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bars , 10 μm .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm.


E , F Meiotic metaphase II spread preparations of Miwi heterozygous ( E ) knockout ( F ) spermatocytes . Scale bars , 5 μm . G Frequency of meiotic metaphase II ( MII ) with chromosomal abnormalities . Chromosome were defined as number of paired sister chromatids per MII metaphase . frequency was calculated as number of with given pairs of sister chromatids ( 20 , < 20 , or > 20 ) divided by total number of . Three mice were used for each genotype , total numbers of counted MII in Miwi heterozygous knockout are 204 302 , respectively .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm. G Frequency of meiotic metaphase II (MII) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired sister chromatids per MII metaphase cell. The frequency was calculated as the number of cells with given pairs of sister chromatids (20, <20, or >20) divided by the total number of cells. Three mice were used for each genotype, total numbers of counted MII cells in Miwi heterozygous and knockout are 204 and 302, respectively.


E , F Meiotic metaphase II spread preparations of Miwi heterozygous ( E ) knockout ( F ) spermatocytes . Scale bars , 5 μm .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm.


H , I Meiotic metaphase I spread preparations of Miwi heterozygous ( H ) knockout ( I ) spermatocytes . Scale bars , 5 μm . J Frequency of meiotic metaphase I ( MI ) with chromosomal abnormalities . Chromosome were defined as number of paired homologous chromosomes per MI metaphase . frequency was calculated in same way as in ( G ) , except for that number of paired chromosomes was counted instead . Three mice were used for each genotype , total numbers of counted MI in Miwi heterozygous knockout are 265 358 , respectively .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm. J Frequency of meiotic metaphase I (MI) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired homologous chromosomes per MI metaphase cells. The frequency was calculated in the same way as in (G), except for that the number of paired chromosomes was counted instead. Three mice were used for each genotype, total numbers of counted MI cells in Miwi heterozygous and knockout are 265 and 358, respectively.


H , I Meiotic metaphase I spread preparations of Miwi heterozygous ( H ) knockout ( I ) spermatocytes . Scale bars , 5 μm .

H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm.


K , L Mitotic metaphase spread preparations of Miwi heterozygous ( K ) knockout ( L ) testes . Scale bars , 5 μm . M Frequency of mitotic metaphase with chromosomal abnormalities . Chromosome were defined as number of chromosomes per mitotic metaphase . Three mice were used for each genotype , total numbers of counted mitotic metaphase in Miwi heterozygous knockout are 52 71 , respectively .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

K, L Mitotic metaphase spread preparations of Miwi heterozygous (K) and knockout (L) testes. Scale bars, 5 μm. M Frequency of mitotic metaphase cells with chromosomal abnormalities. Chromosome counts were defined as the number of chromosomes per mitotic metaphase cell. Three mice were used for each genotype, total numbers of counted mitotic metaphase cells in Miwi heterozygous and knockout are 52 and 71, respectively.


A Equal amounts of total isolated from Miwi knockout ( - / - ) , heterozygote ( + / - ) slicer activity - deficient mutant ( - / ADH ) spermatocytes , were resolved on a 1 % agarose gel . gel was subjected to northern blot using probes specific for sense ( S ) antisense ( As ) strands of major ( Maj ) minor ( Min ) satellite transcripts . blot was re - hybridized with a 28S - complementary probe to demonstrate equal loading ( bottom on left ) . relative abundance was quantified using ImageJ software , normalized to 28S signal from blot .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Equal amounts of total RNA isolated from Miwi knockout (-/-), heterozygote (+/-) and slicer activity-deficient mutant (-/ADH) spermatocytes, were resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite RNA transcripts. The blot was re-hybridized with a 28S-complementary probe to demonstrate equal loading (bottom on the left). The relative abundance was quantified using ImageJ software, normalized to the 28S signal from the blot.


B 100ng of total was isolated from Miwi heterozygous ( + / - ) , knockout ( - / - ) slicer activity - deficient mutant ( - / ADH ) spermatocytes was subjected to qRT - PCR with primers specific to major satellite , minor satellite , 5S , 5 . 8S Gapdh . Data were normalized to 5S rRNA . 5 . 8S rRNA Gapdh were used as negative controls .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B 100ng of total RNA was isolated from Miwi heterozygous (+/-), knockout (-/-) and slicer activity-deficient mutant (-/ADH) spermatocytes and was subjected to qRT-PCR with primers specific to major satellite RNA, minor satellite RNA, 5S, 5.8S and Gapdh. Data were normalized to 5S rRNA. 5.8S rRNA and Gapdh were used as negative controls.


B 100ng of total was isolated from Miwi heterozygous ( + / - ) , knockout ( - / - ) slicer activity - deficient mutant ( - / ADH ) spermatocytes was subjected to qRT - PCR with primers specific to major satellite , minor satellite , 5S , 5 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B 100ng of total RNA was isolated from Miwi heterozygous (+/-), knockout (-/-) and slicer activity-deficient mutant (-/ADH) spermatocytes and was subjected to qRT-PCR with primers specific to major satellite RNA, minor satellite RNA, 5S, 5.8S


Sertoli - spermatogenic co - cultures were transfected with vectors ectopically expressing both strands of major satellite ( Maj , gray bar ) , both strands of minor satellite ( Min , black bar ) , full - 18S ( 18S , gray bar ) or empty vector ( Ctrl , bar ) . Total isolated from equal numbers of FACS - sorted MIWI - expressing spermatocytes were subjected to qRT - PCR with primers specific to major satellite ( C ) , minor satellite ( D ) , 18S ( E )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to major satellite RNA (C), minor satellite RNA (D), 18S (E)


Sertoli - spermatogenic co - cultures were transfected with vectors ectopically expressing both strands of major satellite ( Maj , gray bar ) , both strands of minor satellite ( Min , black bar ) , full - 18S ( 18S , gray bar ) or empty vector ( Ctrl , bar ) . Total isolated from equal numbers of FACS - sorted MIWI - expressing spermatocytes were subjected to qRT - PCR with primers specific to 5 . 8S ( F ) Gapdh ( G ) . Data presented were normalized to 5S rRNA . 5 . 8S rRNA ( F ) Gapdh ( G ) were used as negative controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to 5.8S (F) and Gapdh (G). Data presented were normalized to 5S rRNA. 5.8S rRNA (F) and Gapdh (G) were used as negative controls.


H Immunofluorescence of Meiotic metaphase I plate in Sertoli - spermatogenic co - cultures . Shown were spermatocytes over - expressing both strands of major satellite ( Maj ) , both strands of minor satellite ( Min ) , full - 18S ( 18S ) or empty vector ( Ctrl ) . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

H Immunofluorescence images of Meiotic metaphase I plate in Sertoli-spermatogenic cell co-cultures. Shown were spermatocytes over-expressing both strands of major satellite RNA (Maj), both strands of minor satellite RNA (Min), full-length 18S (18S) or empty vector (Ctrl). Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bar, 5 μm.


I Frequency of mis - arrangement of meiotic metaphase I plate in Sertoli - spermatogenic cocultures using Miwi + / - ; Stra8 - Cre testes . Spermatocytes transfected with vectors expressing both strands of major satellite ( Maj , gray ) , both strands of minor satellite ( Min , black ) , full - 18S ( 18S , gray ) or empty vector ( Ctrl , ) . n = 3 cultures for each group . total numbers of counted meiotic metaphase plate in Ctrl , 18S , Maj Min were 153 , 144 , 130 141 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Frequency of mis-arrangement of the meiotic metaphase I plate in Sertoli-spermatogenic cell cocultures using Miwi+/-;Stra8-Cre testes. Spermatocytes transfected with vectors expressing both strands of major satellite RNA (Maj, dark gray), both strands of minor satellite RNA (Min, black), full-length 18S (18S, light gray) or empty vector (Ctrl, white). n=3 cultures for each group. The total numbers of counted meiotic metaphase plate in Ctrl, 18S, Maj and Min were 153, 144, 130 and 141, respectively.


J Combined - FISH of major satellite transcripts regions in Miwi heterozygote ( Miwi + / - ) knockout ( Miwi - / - ) diakinesis spermatocytes . Sense major satellite transcripts were first detected using an antisense probe ( red signal ) , then major satellite region was further detected using a sense probe ( green signal ) . For RNase A treatment , slides were incubated with ( + RNase A ) or without ( - RNase A ) 100 μg / ml RNase A for 1 hour before adding antisense probe . was stained by DAPI ( blue ) . Arrows indicate same pericentric heterochromatin in each channel per . Scale bars represent 2 µm .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Combined RNA-DNA FISH analysis of major satellite transcripts and DNA regions in Miwi heterozygote (Miwi+/-) and knockout (Miwi-/-) diakinesis spermatocytes. Sense major satellite RNA transcripts were first detected using an antisense probe (red signal), and then the major satellite DNA region was further detected using a sense probe (green signal). For RNase A treatment, the slides were incubated with (+RNase A) or without (-RNase A) 100 μg/ml RNase A for 1 hour before adding antisense probe. Nuclear DNA was stained by DAPI (blue). Arrows indicate the same pericentric heterochromatin in each channel per cell. Scale bars represent 2 µm.


A Combined immunofluorescence FISH of kinetochore inner plate / centromere formation in Miwi heterozygous ( Miwi + / - ) knockout ( Miwi - / - ) spermatocytes at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , are presented in Fig EV1A . Chromosomes were stained with anti - centromere antibody ( CREST , purple signal ) , indicating kinetochore inner plate / centromere . Major satellite regions were detected using an antisense probe ( red signal ) which revealed specific localization of pericentromeres in chromosomes . was stained by DAPI ( blue ) . Scale bars represent 2 µm .

O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Combined immunofluorescence and DNA FISH analysis of the kinetochore inner plate/centromere formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) spermatocytes at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1A. Chromosomes were stained with anti-centromere protein antibody (CREST, purple signal), indicating the kinetochore inner plate/centromere. Major satellite DNA regions were detected using an antisense probe (red signal) which revealed the specific localization of pericentromeres in chromosomes. Nuclear DNA was stained by DAPI (blue). Scale bars represent 2 µm.


B Left panel : illustration of kinetochore configurations in meiotic metaphase I . kinetochores of sister chromatids cohere to form a single microtubule - binding interface . inner - outer kinetochore axis connects pericentric to spindle microtubule . CREST ( centromere ) BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for inner outer regions of kinetochore , respectively . A major satellite ( Maj sat ) probe was used to detect pericentric . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see blue arrow in A ) kinetochores . scale bar represents 1 µm . C Population of overlapping , adjacent , separated inner kinetochores shown in B . results represent mean ± SD of three independent experiments . n = 3 mice for each genotype . total numbers of counted in Miwi + / - , Miwi - / - Miwi - / ADH spermatocytes were 88 , 118 125 , respectively . total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - Miwi - / ADH spermatocytes were 3 , 520 , 4 , 720 5 , 000 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

B Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm. C Population of overlapping, adjacent, and separated inner kinetochores shown in B. The results represent the mean ± SD of three independent experiments. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 88, 118 and 125, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,520, 4,720 and 5,000, respectively.


B Left panel : illustration of kinetochore configurations in meiotic metaphase I . kinetochores of sister chromatids cohere to form a single microtubule - binding interface . inner - outer kinetochore axis connects pericentric to spindle microtubule . CREST ( centromere ) BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for inner outer regions of kinetochore , respectively . A major satellite ( Maj sat ) probe was used to detect pericentric . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see blue arrow in A ) kinetochores . scale bar represents 1 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm.


D Combined immunofluorescence FISH of kinetochore inner outer plate formation in Miwi heterozygous ( Miwi + / - ) knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) anti - centromere - ( CREST , purple ) antibodies to mark kinetochore outer inner plate , respectively . In addition , major satellite regions were further detected using an antisense probe ( red signal ) . was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , blue arrow ; S - O , green arrow . Scale bars represent 2 µm . E Magnified of categories of sister kinetochore configurations from D . One arrow indicates overlapping pair . Double arrows indicate split pair . scale bar represents 1 µm . F Population of sister kinetochores shown in E . n = 3 mice for each genotype . total numbers of counted in Miwi + / - , Miwi - / - Miwi - / ADH spermatocytes were 93 , 89 81 , respectively . total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - Miwi - / ADH spermatocytes were 3 , 720 , 3 , 560 3 , 240 , respectively .

O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm. E Magnified images of categories of sister kinetochore configurations from D. One arrow indicates overlapping pair. Double arrows indicate split pair. The scale bar represents 1 µm. F Population of the sister kinetochores shown in E. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 93, 89 and 81, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,720, 3,560 and 3,240, respectively.


D Combined immunofluorescence FISH of kinetochore inner outer plate formation in Miwi heterozygous ( Miwi + / - ) knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) anti - centromere - ( CREST , purple ) antibodies to mark kinetochore outer inner plate , respectively . In addition , major satellite regions were further detected using an antisense probe ( red signal ) . was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , blue arrow ; S - O , green arrow . Scale bars represent 2 µm .

O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm.


B pull - down using biotinylated major minor satellite transcripts whole extracts from Miwi heterozygoous ( Miwi + / - ) , knockout ( Miwi - / - ) , slicer activity - deficient mutant ( Miwi - / ADH ) , Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . Approximately one repeat of In vitro transcribed major minor satellite RNAs corresponding to sequences in A were used as baits . Precipitated were visualized by Western blotting using antibodies against indicated ( MIWI MILI ) . input equals 1 / 10 to 1 / 80 of lysate used in pull - down . Transcripts corresponding to 300 nt 162 nt of 18S rRNA served as controls for antisense ( As ) sense ( S ) of major minor satellite transcripts , respectively . blot was re - probed with anti - GAPDH antibody serving as a negative control .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B RNA pull-down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous (Miwi+/-), knockout (Miwi-/-), slicer activity-deficient mutant (Miwi-/ADH), and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits. Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins (MIWI and MILI). The input equals 1/10 to 1/80 of the protein lysate used in the pull-down. Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense (As) and sense (S) sequence of major and minor satellite transcripts, respectively. The blot was re-probed with anti-GAPDH antibody serving as a negative control.


C In vitro processing with nuage - fraction from testicular lysates of Miwi knockout ( - / - ) , heterozygous ( + / - ) , slicer activity - deficient ( - / ADH ) mutants , 5 ' - end radioactively labelled targets corresponding to approximately one two repeats of major minor satellite sequences ( see panel A for details ) . Transcripts corresponding to 162 , 300 , 542 nt of 18S rRNA served as controls . Arrow indicates cleavage products of In vitro transcribed sense major satellite RNAs after reactions .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C In vitro RNA processing assay with nuage-nuclear fraction from testicular lysates of Miwi knockout (-/-), heterozygous (+/-), and slicer activity-deficient (-/ADH) mutants, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences (see panel A for details). Transcripts corresponding to 162, 300, and 542 nt of the 18S rRNA sequence served as controls. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions.


D In vitro processing with nuage - fraction from Mov10l1 heterozygous ( Mov10l1f / + ; Stra8 - Cre ) Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testis lysates , 5 ' - end radioactively labelled targets corresponding to approximately one two repeats of sense antisense major satellite sequences . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . arrow indicates cleavage product of in vitro transcribed sense major satellite .

O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

D In vitro RNA processing assay with nuage-nuclear fraction from Mov10l1 heterozygous (Mov10l1f/+;Stra8-Cre) and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testis lysates, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA.


- immunoprecipitation was performed using anti - MIWI antibody . was extracted from anti - MIWI immunoprecipitates subjected to Northern blot using probes specific for potential piRNAs ( piR major - a b ) , as indicated in panel 20 % of input from each group was used as control . blot was re - hybridized with piR - A piR - E complementary probes to demonstrate efficiency of - IP . A miR - 16 complementary probe served as a negative control .

O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

RNA-immunoprecipitation was performed using anti-MIWI antibody. RNA was extracted from anti-MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs (piR major-a and b), as indicated in panel 20% of input RNA from each group was used as the control. The blot was re-hybridized with piR-A and piR-E complementary probes to demonstrate the efficiency of the RNA-IP. A miR-16 complementary probe served as a negative control.


A pull - down was performed using in vitro transcribed biotinylated transcripts whole extracts from Miwi + / - , Miwi - / - , Miwi - / ADH , Mov10l1f / - ; Stra8 - Cre testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . In vitro transcribed RNAs corresponding to approximately one repeat of major minor satellite sequences were used as baits . Precipitated was visualized by Western blotting using anti - Dicer antibody .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

A RNA pull-down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi+/-, Miwi-/-, Miwi-/ADH, and Mov10l1f/-;Stra8-Cre testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits. Precipitated protein was visualized by Western blotting using anti-Dicer antibody.


B Equal amounts of total isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were resolved on a 1 % agarose gel . gel was subjected to Northern blot using probes specific for sense antisense sequences of major minor satellites . blot was re - hybridized with a 28S rRNA - complementary probe to demonstrate equal loading ( bottom on left ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left).


C in vitro processing with nuage - enriched fraction from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre mice , 5 ' - end radioactively labelled targets corresponding to approximately one two repeats of sense antisense major satellite sequences . Arrow indicates cleavage products of In vitro transcribed sense major satellite RNAs by MIWI - piRNA .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

C in vitro RNA processing assay with nuage-enriched nuclear fraction from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, and Miwi+/-;Dicerf/-;Stra8-Cre mice, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI-piRNA complex.


D Equal amounts of total isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were digested with RNase III ( RNase III ) or nuclease S1 ( S1 ) , further resolved on a 1 % agarose gel . gel was subjected to northern blot using probes specific for sense ( S ) antisense ( As ) strands of major ( Maj ) minor ( Min ) satellite transcripts . blot was re - hybridized with 18S 28S - complementary probes to demonstrate equal loading same efficiency of RNase III or nuclease S1 digestion .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion.


E Sequential in vitro processing using 5 ' - end radioactively labelled targets corresponding to approximately one repeat of sense antisense major satellite sequences . nuage - fraction from Miwi + / - testes is indicated as N . Total extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG ( IgG ) as a negative control . incubation time ( Incub . t ) was 0 . 5 1 hour . arrow indicates product of MIWI - mediated cleavage of in vitro transcribed sense major satellite .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG ) as a negative control. The incubation time (Incub. t) was 0.5 and 1 hour. The arrow indicates the product of MIWI-mediated cleavage of the in vitro transcribed sense major satellite RNA.


E Sequential in vitro processing using 5 ' - end radioactively labelled targets corresponding to approximately one repeat of sense antisense major satellite sequences . nuage - fraction from Miwi + / - testes is indicated as N . Total extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O

E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG


A Frequency of overlapping , adjacent , separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A 3B . n = 3 mice for each genotype . total numbers of counted in four types of spermatocytes were 101 , 117 , 129 115 , respectively . total numbers of counted sister kinetochore pairs in four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 4 , 600 , respectively . B Frequency of different sister kinetochore configurations . four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O - O , S - S , O - S , or S - O as defined in text . n = 3 mice for each genotype . total numbers of counted in four types of spermatocytes were 134 , 140 , 140 130 , respectively . total numbers of counted sister kinetochore pairs in four types of spermatocytes were 5 , 360 , 5 , 600 , 5 , 600 5 , 200 , respectively .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively. B Frequency of the different sister kinetochore configurations. The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O-O, S-S, O-S, or S-O as defined in the text. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 134, 140, 140 and 130, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5,360, 5,600, 5,600 and 5,200, respectively.


A Frequency of overlapping , adjacent , separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A 3B . n = 3 mice for each genotype . total numbers of counted in four types of spermatocytes were 101 , 117 , 129 115 , respectively . total numbers of counted sister kinetochore pairs in four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 4 , 600 , respectively .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively.


C Frequency of abnormal arrangement in meiotic metaphase I plate . four types of spermatocytes in A B were used to perform immunofluorescence as shown in Fig 1B C . n = 3 mice for each genotype . total numbers of counted metaphase I plates in four types of spermatocytes were 221 , 181 , 421 240 , respectively . D Frequency of meiosis I metaphase with chromosomal abnormalities . four types of spermatocytes in A - C were used to perform a metaphase spread as shown in Fig 1H 1I . n = 3 mice for each genotype . total numbers of counted MI in four types of spermatocytes were 89 , 73 , 132 104 , respectively . E Frequency of meiosis II metaphase with chromosomal abnormalities . four types of spermatocytes in A - D were used to perform a metaphase spread as shown in Fig 1K L . n = 3 mice for each genotype . total numbers of counted MII in four types of spermatocytes were 74 , 87 , 85 94 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively. D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively. E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.


C Frequency of abnormal arrangement in meiotic metaphase I plate . four types of spermatocytes in A B were used to perform immunofluorescence as shown in Fig 1B C . n = 3 mice for each genotype . total numbers of counted metaphase I plates in four types of spermatocytes were 221 , 181 , 421 240 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.


A - F ' Upper panels ( A - F ) show of c4da neurons of indicated genotypes at third instar larval stage , lower panels ( A ' - F ' ) show c4da neuron at 18 h after puparium formation ( APF ) . Neurons were labeled by CD8 : : GFP expression under ppk - GAL4 or by tdtomato expression in MARCM clones . ( A , A ' ) C4da neurons expressing control Orco dsRNA . Scale bars are 100 μm in A ( for larval ) 50 μm in A ' ( for pupal ) . ( B , B ' ) C4da neurons expressing PP2A - 29B dsRNA . ( C , C ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones . ( D , D ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones expressing UAS - HAPP2A - 29B . ( E , E ' ) C4da neurons expressing dominant - negative Mts . ( F , F ' ) Homozygous mtss5286 mutant c4da neuron MARCM clones . G Penetrance of pruning defects at 18 h APF in A ' - E ' . * * P < 0 . 005 , * * * P < 0 . 0005 , Fisher ' s exact . Number of neurons for each genotype are given in figure , number of is given below in parentheses . H Severity of pruning defects at 18 h APF in A ' - E ' as assessed by number of secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s . Numbers of neurons ( ) for each genotype are given in figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - J indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , dendrite pruning defects were assessed at 18 h APF . Panels A - E show effects of Sox14 / Mical pathway manipulations alone , panels F - J show neurons coexpressing PP2A - 29B dsRNA . scale bar in A is 50 μm . ( A , F ) C4da neurons ( co - ) expressing lacZ . ( B , G ) C4da neurons ( co - ) overexpressing Sox14 . ( C , H ) C4da neurons ( co - ) overexpressing Mical . ( D , I ) C4da neurons ( co - ) expressing Mical ∆ redox . ( E , J ) C4da neurons ( co - ) expressing Mical ∆ CH . K Penetrance of pruning defects in panels A - J . * P < 0 . 05 , * * * P < 0 . 0005 , Fisher ' s exact . Numbers of neurons ( ) for each genotype are given in figure numbers of are given below in parentheses . L Severity of pruning defects in panels A - J as assessed by number of secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * * P < 0 . 0005 , Wilcoxon ' s . Numbers of neurons ( ) for each genotype are given in figure .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , expression of Sox14 was assessed by immunofluorescence . A - C Sox14 stainings at 2 h APF . ( A ) Control c4da neurons . ( B ) C4da neurons expressing PP2A - 29B dsRNA . ( C ) C4da neuron expressing dominant - negative Mts . D of Sox14 expression in ( A - C ) . C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons ( identified by characteristic shape ) . Data are mean + / - s . d . . numbers of neurons ( ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * * P < 0 . 0005 , Wilcoxon ' s .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , expression of Mical was assessed by immunofluorescence . E - H Mical stainings at indicated time points . ( E ) Mical expression in control c4da neurons at 2 h APF . ( F ) Mical expression in c4da neurons expressing dominant - negative Mts at 2 h APF . ( G ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 2 h APF . ( H ) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF . ( I ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 0 h APF . J of Mical expression in ( E - I ) . C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons ( marked by asterisks ) . Data are mean + / - s . d . . numbers of neurons ( ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s . Data information : Scale bars in A E are 10 μm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.


A - G indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression were performed as in Figure 2 . Panels A - C show effects of expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . arrow in panel G denotes a characteristic varicosity at tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA lacZ as a UAS titration control . H Penetrance of pruning defects in panels A - G . * P < 0 . 05 , Fisher ' s exact . Numbers of neurons ( ) for each genotype are given in figure . I Severity of pruning defects in panels A - G as assessed by number of secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , Wilcoxon ' s . Numbers of neurons ( ) for each genotype are given in figure .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control. H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - G indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression were performed as in Figure 2 . Panels A - C show effects of expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . arrow in panel G denotes a characteristic varicosity at tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA lacZ as a UAS titration control .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control.


J - L GFP - tagged Drosophila cofilin ( GFP : : tsr ) was expressed in c4da neurons under ppk - GAL4 , its localization was assessed at indicated developmental stages by live imaging . Panels J - L show GFP : : tsr localization in c4da neurons at third instar stage , panels J ' - L ' show GFP : : tsr localization in c4da neurons at 6 h APF . ( J , J ' ) Control c4da neurons expressing Orco dsRNA . ( K , K ' ) C4da neurons expressing Mical dsRNA . ( L , L ' ) C4da neurons expressing PP2A - 29B dsRNA . M of GFP : : tsr in samples J - L ' . Data are mean + / - s . d . , numbers of neurons ( ) analyzed for each genotype are given in figure ( data were not derived from independent experiments ) . * * P < 0 . 005 , * * * P < 0 . 0005 , n . s . , not significant , Wilcoxon ' s .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA. M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.


A - H Genetic interactions between PP2A - 29B actin regulators during dendrite pruning . indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression were performed as in Figure 2 . ( A ) C4da neuron coexpressing PP2A - 29B dsRNA tdtomato as suppression control . ( B ) C4da neuron coexpressing PP2A - 29B dsRNA GFP : : tsr . ( C ) C4da neuron coexpressing PP2A - 29B dsRNA RacN17 . ( D ) C4da neuron coexpressing PP2A - 29B dsRNA RhoN19 . ( E ) C4da neuron expressing PP2A - 29B dsRNA in ena210 / + background . ( F ) C4da neuron coexpressing PP2A - 29B dsRNA Orco dsRNA as suppression control . ( G ) C4da neuron coexpressing PP2A - 29B dsRNA Chicadee dsRNA . ( H ) C4da neuron coexpressing PP2A - 29B dsRNA moesin dsRNA . I Penetrance of pruning defects in panels E - L . n . s . , not significant , Fisher ' s exact . Numbers of neurons ( ) for each genotype are given in figure . J Severity of pruning defects in panels E - L as assessed by number of secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s . Numbers of neurons ( ) for each genotype are given in figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A . Immunohistochemical of SHARPIN expression in human mammary . Cross - section of a mammary ( upper panel ) magnification of marked ( lower panel ) . SHARPIN - positive luminal ( grey arrow ) stromal ( red arrow ) , approximate position of basal lamina ( dashed red ) are indicated . Scale bar represents 50 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm.


B . Western blot of SHARPIN expression in isolated mammary epithelial ( MECs ) mammary stromal fibroblasts ( MSFs ) . CDH1 vimentin were used as markers of epithelial stromal lineages , respectively . GAPDH served as a control for loading .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading.


C . FACS - isolation of mouse mammary basal epithelial ( LinnegCD24intICAM - 1hi ) , mature luminal epithelial ( LinnegCD24hiICAM - 1neg ) , luminal progenitor ( LinnegCD24hiICAM - 1int ) , stromal ( LinnegCD24neg ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O

C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg).


D . Quantitative PCR of SHARPIN mRNA expression in populations isolated in ( C ) ( mean ± , n = 3 - 5 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).


A - G . Mammary outgrowth in 3 - 7 old wt or Sharpincpdm mice . A . Representative carmine - alum stained mammary whole mounts . Arrow indicates inguinal lymph node . Scale bars represent 2 mm . B . of mammary outgrowth ( n = 4 - 15 ) . C . of number of branch points per mammary ( n = 7 - 9 , 7 old mice ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice).


A - G . Mammary outgrowth in 3 - 7 old wt or Sharpincpdm mice . D . Cryosections from 7 old wt or Sharpincpdm mouse mammary stained with HE ( upper panel ) or immunolabelled with indicated antibodies . Scale bars represent 20

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm


A - G . Mammary outgrowth in 3 - 7 old wt or Sharpincpdm mice . E . Representative carmine - alum stained highlighting terminal end buds ( TEBs ) in 7 old wt Sharpincpdm mouse mammary F . of number of TEBs per ( n = 9 - 10 ) . Scale bar represents 500 μm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm.


A - G . Mammary outgrowth in 3 - 7 old wt or Sharpincpdm mice . G . TEBs in paraffin sections from wt or Sharpincpdm mouse mammary stained with HE ( upper panel ) or immunolabelled with indicated antibodies ( lower panel ) . Scale bars represent 50 µm . Mean ± .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.


A - C . Monitoring of mammary development following transplantation of wt or Sharpincpdm mouse mammary epithelium in mammary fat pads ( epithelium - free ) of virgin wt mice . A . Representative carmine - alum stained of mouse mammary generated from wt or Sharpincpdm epithelial tissue transplants . B . of transplant growth take - on - rate ( n = 17 ) . C . of fat pad filling rate of grown transplants ( n = 7 - 10 ) in wt hosts .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. A. Representative carmine-alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. B. Quantification of transplant growth take-on-rate (n = 17). C. Quantification of fat pad filling rate of grown transplants (n = 7 - 10) in wt hosts.


D . Monitoring of mammary differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts . Host were subsequently mated mammary isolated at P15 . Representative Carmine - alum stained mammary whole mounts generated from wt cdpm mammary epithelial transplants ( upper panel ) , magnifications of branched epithelium ( lower panel ) are shown ( n = 5 mice ) . Scale bars represent 1 mm .

D. Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts. Host animals were subsequently mated and mammary glands isolated at P15. Representative Carmine-alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants (upper panel), and magnifications of the branched ductal epithelium (lower panel) are shown (n = 5 mice). Scale bars represent 1 mm.


E . Representative of carmine - alum stained mouse mammary whole mounts generated from 7 old S100a4 - Cre ; Sharpinfl / fl conditional knockout mice their littermate controls ( S100a4 - Cre ; Sharpin fl / + ) F . of normalized Sharpincpdm S100a4 - Cre ; Sharpinfl / fl mammary outgrowth relative to littermate control mice ( wt Sharpincpdm n = 10 ; S100a4 - Cre ; Sharpin fl / + S100a4 - Cre ; Sharpinfl / fl n = 8 ) . G . of number of TEBs per ( n = 8 - 9 ) . Mean ± .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative images of carmine-alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4-Cre; Sharpinfl/fl conditional knockout mice and their littermate controls (S100a4-Cre; Sharpin fl/+) F. Quantification of the normalized Sharpincpdm and S100a4-Cre; Sharpinfl/fl mammary ductal outgrowth area relative to littermate control mice (wt and Sharpincpdm n = 10 glands; S100a4-Cre; Sharpin fl/+ and S100a4-Cre; Sharpinfl/fl n = 8 glands). G. Quantification of the number of TEBs per gland (n = 8 - 9 glands). Mean ± SEM.


A . Second harmonic generation ( SHG ) unfiltered multiphoton ( MP ) imaging of collagen fibres surrounding TEBs ( dashed ) in 6 - 7 old wt Sharpincpdm mouse mammary ( n = 6 - 9 mice , 5 - 13 TEBs analysed per mouse ; scale bars represent 100 µm ) . Magnified of collagen fibres are also shown ( scale bars represent 50 µm ) . B . Frequency of clear collagen bundles around TEBs per mouse ( Mean ± , n = 6 - 9 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Second harmonic generation (SHG) and unfiltered multiphoton (MP) imaging of collagen fibres surrounding TEBs (dashed line) in 6-7 weeks old wt and Sharpincpdm mouse mammary glands (n = 6 - 9 mice, 5 - 13 TEBs analysed per mouse; scale bars represent 100 µm). Magnified images of collagen fibres are also shown (scale bars represent 50 µm). B. Frequency of clear collagen bundles around TEBs per mouse (Mean ± SEM, n = 6 - 9 mice).


C . AFM indentation of stromal stiffness in 6 - 7 old wt Sharpincpdm mouse mammary tissue sections ( n = 3 mice ; 3 - 6 sample sections per mouse 192 indentation curves per sample ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

C. AFM indentation analysis of stromal stiffness in 6-7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections (n = 3 mice; 3 - 6 sample sections per mouse and 192 indentation curves per sample).


A . Ingenuity Pathway of differential gene expression ( fold - change ( FC ) > 1 . 5 , p < 0 . 05 ) in wt Sharpincpdm MSFs ( passage 0 ) . differentially expressed genes implicated in most significantly altered canonical pathways ( leukocyte adhesion transmigration , tissue fibrosis ) are shown . Three independent isolation replicates were compared pairwise . FC colour scale represents a range from 0 − 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Ingenuity Pathway Analysis of differential gene expression (fold-change (FC) > 1.5, p < 0.05) in wt and Sharpincpdm MSFs (passage 0). The differentially expressed genes implicated in the most significantly altered canonical pathways (leukocyte adhesion and transmigration, and tissue fibrosis) are shown. Three independent cell isolation replicates were compared pairwise. FC colour scale represents a range from 0−4.


B . of collagen degradation on cross - bow - shaped micropatterns coated with DQ collagen substrate . Mean projection of DQ collagen ( top panel ; is outlined by dashed ; original integer range 0 − 255 ) single confocal of fibrinogen - Alexa647 - marked micropatterns ( bottom panel ) are shown ( n ≥ 53 from four independent experiments ) . Scale bars represent 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Analysis of collagen degradation on cross-bow-shaped micropatterns coated with the DQ collagen substrate. Mean intensity projection images of the DQ collagen fluorescence (top panel; cell area is outlined by dashed line; original integer range 0 − 255) and single confocal images of the fibrinogen-Alexa647-marked micropatterns (bottom panel) are shown (n ≥ 53 from four independent experiments). Scale bars represent 10 μm.


C - D . Collagen plug contraction using wt Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C . Representative microscopy of collagen plugs three days post culture . D . of average collagen plug 1 − 3 days after seeding ( n = 3 - 6 ) . Scale bars represent 2 mm .

O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C. Representative light microscopy images of the collagen plugs three days post cell culture. D. Quantification of the average collagen plug area 1−3 days after seeding (n = 3-6). Scale bars represent 2 mm.


E . SHARPIN level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs . Tubulin was used as loading control .

O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

E. SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs. Tubulin was used as loading control.


F - I . Traction force microscopy of wt Sharpincpdm MSFs on collagen - coated matrices . F . Representative of traction forces observed in wt Sharpincpdm ( colour scale units : Pa ; boundaries are indicated by dashed lines ) . G . of energy per ( n = 8 − 11 from two independent experiments ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments).


F - I . Traction force microscopy of wt Sharpincpdm MSFs on collagen - coated matrices . H . Representative of traction forces mCherry of mCherry - Ctrl transfected wt mCherry - Ctrl or mCherry - SHARPIN transfected Sharpincpdm . Scale bars represent 20 µm . I . of energy per ( wt mCherry control n = 26 ; Sharpincpdm mCherry - Ctrl n = 21 ; Sharpincpdm mCherry - SHARPIN n = 24 from five independent experiments ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O

F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. H. Representative images of the traction forces and mCherry fluorescence of mCherry-Ctrl transfected wt cells and mCherry-Ctrl or mCherry-SHARPIN transfected Sharpincpdm cells. Scale bars represent 20 µm. I. Quantification of strain energy per cell (wt mCherry control n = 26; Sharpincpdm mCherry-Ctrl n = 21; Sharpincpdm mCherry-SHARPIN n = 24 cells from five independent experiments).


J - K . Focal adhesion dynamics . J . Visualization of GFP - Paxillin dynamics in a wt a Sharpincpdm over time . Colour scale represents a range from early to late occurring focal adhesions . K . of focal adhesion average , assembly rate disassembly rate ( wt n = 17527 , 5250 , 5311 FAs ; Sharpincpdm n = 9052 , 2790 , 3264 FAs , respectively from two independent experiments ) . Mean ± .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

J-K. Focal adhesion dynamics. J. Visualization of GFP-Paxillin dynamics in a wt and a Sharpincpdm cell over time. Colour scale represents a range from early to late occurring focal adhesions. K. Quantification of focal adhesion average size, assembly rate and disassembly rate (wt n =17527, 5250, 5311 FAs; and Sharpincpdm n= 9052, 2790, 3264 FAs, respectively from two independent experiments). Mean ± SEM.


A . of soluble collagen in wt Sharpincpdm MSF cultures on days 2 , 5 7 post serum removal ( n = 6 from two independent experiments ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

A. Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2, 5 and 7 post serum removal (n = 6 from two independent experiments).


B - C . of collagen network in - derived matrices ( CDMs ) produced by wt Sharpincpdm MSFs using COL1A1 immunolabelling . B . Maximum projections of confocal are shown . Scale bars represent 50 μm . C . of distinct collagen fibre bundles in wt Sharpincpdm CDM samples ( n = 5 ) . Mean ± .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

B-C. Analysis of the collagen network in cell-derived matrices (CDMs) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling. B. Maximum intensity projections of confocal images are shown. Scale bars represent 50 μm. C. Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples (n = 5). Mean ± SEM.


were fixed viral bait fused with V5 - tag was visualized by immunofluorescence using Alexa Fluor - 488 labeled anti - V5 immunostaining ( yellow ) , actin with Alexa Fluor594 - labeled phalloidin ( magenta ) , nuclei were stained with DAPI ( cyan ) ( Scale bar : 10 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were fixed and the viral bait fused with the V5-tag was visualized by immunofluorescence staining using Alexa Fluor-488 labeled anti-V5 immunostaining (yellow), actin with Alexa Fluor594-labeled phalloidin (magenta), and nuclei were stained with DAPI (cyan) (Scale bar: 10 μm).


A For image - drug screening , Calu - 1 were infected with SARS - CoV - 2 in presence of drugs with indicated doses for 48 h , immunostained , imaged analyzed . example of mock - infected - infected control ( no drugs ) . viral yield in was detected with an antibody for viral N ( - infected ; upper image ) , ( mock - infected ; lower image ) ( Scale bar : 200 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A For the image-based drug screening, Calu-1 cells were infected with SARS-CoV-2 in the presence of drugs with indicated doses for 48 h, immunostained, imaged and analyzed. The example images of mock-infected cells and virus-infected control cells (no drugs). The viral yield in cells was detected with an antibody for the viral N protein (virus-infected cells; upper image), and nuclear staining (mock-infected cells; lower image) (Scale bar: 200 μm).


B dose - response curves for six drugs with potential antiviral effects . drug - induced inhibition of infection ( of N positive ) was quantified using under curve ( AUC ) values .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

B The dose-response curves for six drugs with potential antiviral effects. The drug-induced inhibition of virus infection (count of N protein positive cells) was quantified using the area under the curve (AUC) values.


C effect of drugs on viability in presence of , e . g . , cytotoxicity was evaluated by of nuclei .

O O O O O O O O O O O O O O O O O O O O O O

C The effect of drugs on cell viability in the presence of the virus, e.g., cytotoxicity was evaluated by the count of nuclei.


D Inhibition of interactions formed with DDX39B in presence of MTX using co - IP . DDX39B is fused to C terminus of a V5 tag , interaction partner is fused with a Strep - HA tag . diagram ( upper ) shows interaction pairs formed with DDX39B used for co - IP . Dot - blot results ( lower ) show interaction pairs ( arrangement as upper panel ) in presence of different of MTX as indicated on top of image . image displayed is representative of 3 replicates ( n = 3 ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3).


E of DDX39B binding to different interaction partners . of V5 - tagged DDX39B amount was normalized over HA - tagged interaction partner displayed as a fold change over of untreated . Data that represent mean of 3 replicates are highlighted in red . A fold change > 0 . 5 is considered significant .

O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.


A αSMA mRNA in TGFβ - treated HFL1 fibroblasts effect of IRE1α inhibitor 4μ8C . * P = 0 , 001 .

O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.


B Collagen1α2 mRNA in TGFβ - treated HFL1 fibroblasts ± IRE1α inhibitor 4μ8C . * P = 0 , 01 .

O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.


C mRNA of ER - stress related genes in TGFβ - treated HFL1 fibroblasts . * P CHOP = 0 , 045 , * P Bip = 0 , 023 , * P spliced XBP - 1 P = 0 , 001 , compared to corresponding control .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.


D Western blot of CHOP in TGFβ - treated HFL1 ± IRE1α inhibitor 4μ8C .

O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O

D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.


E Agarose gel showing presence of spliced unspliced XBP - 1 mRNA in TGFβ - treated HFL1 fibroblasts ± IRE1α inhibitor 4μ8C .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O

E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.


F Immunohistochemistry of αSMA in TGFβ - treated HFL1fibroblasts ± IRE1α inhibitor 4μ8C . Scale bars = 20μm .

O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

F Immunohistochemistry staining of αSMA in TGFβ-treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.


G of secreted soluble collagen produced by TGFβ - treated HFL1 ± IRE1α inhibitor 4μ8C . * P = 0 , 049 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.