( F ) Immunoprecipitation of DNP52 - RNF34 interaction in infected with VSV . Anti - DNP52 immunoprecipitates were analyzed using immunoblotting with an anti - DNP52 or anti - RNF34 antibody . WCL was immunoblotted with an anti - MAVS or anti - RNF34 antibody . - studies were performed independently at least three times with comparable results .

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(F) Immunoprecipitation analysis of the DNP52-RNF34 interaction in cells infected with VSV. Anti-DNP52 immunoprecipitates were analyzed using immunoblotting with an anti-DNP52 or anti-RNF34 antibody. The WCL was immunoblotted with an anti-MAVS or anti- RNF34 antibody. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoprecipitation of interaction between Flag - NDP52 HA - RNF34 in WT or MAVS - / - . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA antibody . - studies were performed independently at least three times with comparable results .

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(G) Immunoprecipitation analysis of the interaction between Flag-NDP52 and HA-RNF34 in WT or MAVS-/- cells. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA antibody. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoprecipitation of HEK293T transfected with Flag - NDP52 or its D439R / C443K mutant together with HA - MAVS followed by VSV infection . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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(H) Immunoprecipitation analysis of HEK293T cells transfected with Flag-NDP52 or its D439R/C443K mutant together with HA-MAVS followed by VSV infection. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( I ) Immunoblot showing of MAVS in siCtrl - or siNDP52 - transfected expressing Flag - RNF34 or Flag - V . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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(I) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells expressing Flag-RNF34 or Flag-V. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoblot showing of MAVS in siCtrl - or siNDP52 - transfected infected with VSV ( MOI = 1 , 12 hrs ) . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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(J) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells infected with VSV (MOI = 1, 12 hrs). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( K ) Immunoprecipitation of MAVS - LC3 interaction in siCtrl - or siNDP52 - transfected infected with VSV . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - LC3 antibody . NDP52 were analyzed by performing immunoblotting with an anti - NDP52 antibody . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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(K) Immunoprecipitation analysis of the MAVS-LC3 interaction in siCtrl- or siNDP52-transfected cells infected with VSV. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-LC3 antibody. NDP52 levels were analyzed by performing immunoblotting with an anti-NDP52 antibody. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing MAVS , IRF3 - p , LC3 , TOM20 HSP60 in siCtrl - or siRNF34 - 1 infected with VSV cultured in presence of Z - VAD ( 50 μM ) for indicated times . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

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(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing MAVS , LC3 , TOM20 HSP60 in transfected with Flag - RNF34 or its H342A mutant subsequently infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

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(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


Representative confocal ( C ) of immunofluorescence for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 infected with VSV for indicated times . Red represents MAVS green represents NDP52 . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

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Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( D ) of immunofluorescence for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 infected with VSV for indicated times . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

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quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( E ) Transmission electron microscopy of shCtrl - shRNF34 - 1 HEK293T cultured in presence or absence of VSV . Arrows indicate mitochondria engulfed in autophagosomes . Double arrows indicate swollen mitochondria with disrupted cristae . M , mitochondria N , nucleus . 40000 × magnification . - studies were performed independently at least three times with comparable results .

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(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results.


( F ) Representative confocal of shift in emission of Keima from 458 nm to 543 nm . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

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(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


Colony formation of miRNA mimic transfected - Dgcr8 - / - ESCs . There were 17 miRNAs that decreased colony forming ability , 15 miRNAs that did not affect colony formation 8 miRNAs enhanced colony formation of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S1 .

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Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.


Alkaline phosphatase of ESCs after miRNA mimic transfection . There were 21 miRNAs that decreased AP activity , 11 miRNAs that mildly affected AP activity 8 miRNAs boosted AP activity of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S2 .

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Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.


cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8 - / - ESCs . Error bars indicate s . d . ( n = 3 ) . Full data are shown in Supplementary Fig S3A , B .

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Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.


Immunofluorescence of Oct4 in miRNA mimic transfected ESCs . 20 miRNAs decreased Oct4 expression produced small grossly differentiated colonies , 12 miRNAs mildly affected Oct4 expression , 8 miRNAs improved Oct4 expression yielded compact undifferentiated colonies . Representative pictures are shown . Full data are shown in Supplementary Fig S3C .

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Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.


AP of V6 . 5 ESCs after mimic transfection ( Full data are shown in Supplementary Fig S4B ) .

AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).


B , C . Northern blot of expression of miR - 23a , miR - 27a miR - 24 during EB formation ( B ) RA induced differentiation of V6 . 5 ESCs ( C ) . was representive of results of quantitative .

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B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.


D , E . expression of miR - 23a ~ 27a ~ 24 - 2 transcript during EB formation ( D ) RA induced differentiation ( E ) . Error bars indicate s . d . ( n = 3 ) .

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D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).


F . Q - PCR of miR - 23a , miR - 27a miR - 24 expression in ESCs ( JM8A3 ) a variety of C57BL / 6J adult tissues . Data are shown as mean + s . d . ( n = 3 ) .

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F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).


G . Relative of Oct4 , Sox2 Nanog in miRNA mimic transfected Dgcr8 - / - ESCs wild - type ESCs .

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G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.


H , I . Oct4 , Nanog , Foxa2 , Gata2 Nestin immunofluorescence of mimic transfected Dgcr8 - / - ( H ) wild - type ESCs ( I ) .

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H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).


Western blot of expression of target genes in miR - 27a or miR - 24 over - expressed V6 . 5 ESCs . Candidate target genes whose expression was decreased were framed .

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Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.


relative luciferase activity of reporter constructs co - transfected with scramble or miR - 27a miR - 24 mimics . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 . Candidate target genes whose luciferase activity was decreased were framed . PC group was positive reporter control which harbored reverse complementary of miR - 27a or miR - 24 .

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The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.


relative luciferase activity of wild - type 3 ′ UTR or mutated 3 ′ UTR reporter co - transfected with scramble or miR - 27a miR - 24 mimics . Data are shown as mean + s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.


F , G RT - PCR qRT - PCR of in vivo binding of miRNAs with their targets . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


H . Western blot of c - Myc expression in siRNA treated V6 . 5 ESCs .

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H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.


I . Q - PCR of - 23a ~ 27a ~ 24 - 2 , miR - 23a , miR - 27a miR - 24 expression in c - Myc knocked down ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


K . ChIP - PCR ChIP - q - PCR of c - Myc binding to putative sites in promoter . Error bars indicate s . d . ( n = 3 ) ; * * * indicates P - value < 0 . 001 .

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K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.


L . Relative luciferase activity of wild - type - 15 binding site mutated promoter in NIH3T3 co - transfected with c - Myc or blank vector ( NC ) . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


M . Relative luciferase activity of wild - type - 15 binding site mutated promoter in V6 . 5 ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


Validation of effect of miRNA inhibition in MEFs by q - PCR . Error bars indicate s . d . ( n = 3 ) .

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Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).


AP of iPSC colonies in reprogramming with OSK combined with scramble control , inhibitors of miR - 23a , miR - 27a , miR - 24 let - 7c respectively .

AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.


Fold increase in Oct4 - GFP - positive colonies in above . Data are shown as mean + s . d . ( n = 5 ) ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.


of typical Oct4 - GFP - positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors .

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Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.


Detection of endogenous exogenous Oct4 , Sox2 , Klf4 Nanog expression in iPSCs , ESCs MEFs by RT - PCR .

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Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.


Immunofluorescence of Oct4 , Nanog SSEA1 of iPSC colonies .

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Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.


Q - PCR of expression of self - renewal ( Oct4 , Sox2 , Nanog ) differentiation ( Cdx2 , Foxa2 , Brachyury , Hand1 , Nestin , FGF5 ) relative markers in EBs derived from iPSCs . Error bars indicate s . d . ( n = 3 ) .

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Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).


Hematoxylin eosin of teratoma derived from iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) respiratory epithelium ( Endoderm ) are marked with black arrows .

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Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.


Genomic PCR of specific fragments spanning miR - 23 ~ 27 ~ 24 clusters . WT indicate wild - type ESCs ; DKO indicate miR - 23a ~ 27a ~ 24 - 2 - / - , miR - 23b ~ 27b ~ 24 - 1 - / - ESCs ; KO indicate miR - 23a ~ 27a ~ 24 - 2 - / - ESCs .

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Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.


Real - time PCR of mature miR - 23a / b , miR - 27a / b miR - 24 expression in wild - type , DKO KO ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).


AP of wild - type miR - 23 ~ 27 ~ 24 cluster knockout ESCs .

AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.


Real - time PCR of Oct4 , Sox2 Nanog expression in wild - type miR - 23 ~ 27 ~ 24 cluster knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).


Oct4 , Sox2 Nanog immunofluorescence of wild - type knockout ESCs .

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Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.


Real - time PCR of Oct4 several lineage differentiation markers expression during EB formation of wild - type knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Beating EB incidence during cardiac differentiation of wild - type knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Real - time PCR of several cardiac markers expression during cardiac differentiation of wild - type knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Actinin , ANP Troponin Iimmunofluorescence of differentiation cultures derived from wild - type knockout ESCs .

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Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.


Volumes of teratomas at different days after injection . Data are shown as mean + s . d . ( n = 4 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


of teratomas derived from wild - type knockout ESCs . Data are shown as mean ± s . d . * * indicates P - value < 0 . 01 .

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Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.


Oct4 immunohistochemistry of teratomas derived from wild - type knockout ESCs . Representative Oct4 positive regions are shown .

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Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.


Hematoxylin eosin of teratomas derived from wild - type knockout ESCs . Ectoderm , Mesoderm Endoderm are marked with different color arrows .

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Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.


( A ) list of co - precipitated as determined by immunoprecipitation followed by mass spectrometry ( IP - MS ) . SWR1 subunits not shared by ISWI complexes are shown in blue ; DDT domain - containing ISWI specific subunits are shown in orange ; CHR11 CHR17 are shared subunits of SWR1 ISWI complexes are shown in green .

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(A) The list of co-precipitated proteins as determined by immunoprecipitation followed by mass spectrometry (IP-MS). The SWR1 subunits not shared by the ISWI complexes are shown in blue; The DDT domain-containing ISWI specific subunits are shown in orange; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green.


( C ) Determination of interaction of MBD9 with HTA9 , HTA11 , PIE1 , CHR11 , CHR17 by co - immunoprecipitation .

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(C) Determination of the interaction of MBD9 with HTA9, HTA11, PIE1, CHR11, and CHR17 by co-immunoprecipitation.


Determination of interaction of PIE1 - Flag , MBD9 - Myc , CHR11 - HA by in vitro pull down assays . PIE1 - Flag , MBD9 - Myc , CHR11 - HA were expressed from yeast subjected to pull down assays . Anti - Flag agaroses were independently used in pull down assays .

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Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag agaroses were independently used in the pull down assays.


C ) Determination of interaction of MBD9 - Myc , CHR11 - HA by in vitro pull down assays . MBD9 - Myc , CHR11 - HA were expressed from yeast subjected to pull down assays . anti - HA ( C ) agaroses were independently used in pull down assays .

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C) Determination of the interaction of MBD9-Myc, and CHR11-HA by in vitro pull down assays. MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. anti-HA (C) agaroses were independently used in the pull down assays.


D ) he effect of pie1 mbd9 - 1 mutations on formation of SWR1 as determined by IP - MS . Transgenic expressing Flag - tagged MBD9 , PIE1 , CHR11 in WT mutant backgrounds were subjected to IP - MS experiment with anti - Flag agarose .

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D) he effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose.


( A ) Morphological of WT , chr11 / 17 , pie1 , swc2 , rin2 mutant . Shown are 24 - day - old . top panel shows of in soil ; bottom panel shows of on black cloth .

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(A) Morphological phenotypes of the WT, chr11/17, pie1, swc2, and rin2 mutant plants. Shown are 24-day-old plants. The top panel shows the phenotypes of plants in the soil; the bottom panel shows the phenotypes of plants on the black cloth.


( B ) Shown are rosette leaves of WT , pie1 , swc2 , rin2 mutant . Rosette leaves were cut from bolting .

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(B) Shown are rosette leaves of the WT, pie1, swc2, and rin2 mutant plants. Rosette leaves were cut from bolting plants.


( C ) early - flowering phenotype of mbd9 - 1 , mbd9 - 3 , mbd9 - 4 hta9 - 1 / 11 - 1 mutants compared to WT . 24 - day - old in soil are shown .

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(C) The early-flowering phenotype of the mbd9-1, mbd9-3, mbd9-4 and hta9-1/11-1 mutants compared to the WT. 24-day-old plants in soil are shown.


( D ) Numbers of rosette leaves of WT mutant shortly after bolting . Rosette leaves from at least 20 were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

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(D) Numbers of rosette leaves of the WT and mutant plants shortly after bolting. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( E ) Determination of effect of arp4 - 1 , arp6 swc6 - 1 mutations on flowering time . top panel shows number of rosette leaves in WT , arp4 - 1 , arp6 , swc6 - 1 mutant ; bottom panel shows a photograph of . Rosette leaves from at least 20 were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

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(E) Determination of the effect of the arp4-1, arp6 and swc6-1 mutations on flowering time. The top panel shows the number of rosette leaves in the WT, arp4-1, arp6, and swc6-1 mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( F ) Determination of effect of tra1a tra1b mutations on flowering time . top panel shows numbers of rosette leaves of WT , tra1a - 1 , tra1a - 2 , tra1b mutant ; bottom panel shows a photograph of . Rosette leaves from at least 20 null were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

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(F) Determination of the effect of the tra1a and tra1b mutations on flowering time. The top panel shows numbers of rosette leaves of the WT, tra1a-1, tra1a-2, and tra1b mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 null were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( B ) Heat maps showing effect of indicated mutations on expression of differentially expressed genes identified in mbd9 - 1 mutant . color bar shows log2 ( fold change ) . Up - down - regulation are shown by red blue , respectively .

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(B) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9-1 mutant. The color bar shows log2(fold change). Up- and down-regulation are shown by red and blue, respectively.


( C ) Scatter plots showing that expression pattern in hta9 - 1 / 11 - 1 mutant is correlated with that in chr11 / 17 , mbd9 - 1 , pie1 mutants . differentially expressed genes identified in hta9 - 1 / 11 - 1 mutant were subjected to .

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(C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.


( A ) effect of indicated mutations on H2A . Z deposition as determined by immunoblotting . - associated were extracted subjected to immunoblotting by anti - H2A . Z antibody . histone H4 level was detected by anti - H4 antibody indicated as a loading control .

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(A) The effect of indicated mutations on H2A.Z deposition as determined by immunoblotting. Chromatin-associated proteins were extracted and subjected to immunoblotting by the anti-H2A.Z antibody. The histone H4 level was detected by the anti-H4 antibody and indicated as a loading control.


( B ) Meta plots showing H2A . Z occupancy on H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . RPKM , reads per kilobase per million . H2A . Z ChIP - seq results were generated from two independent biological replicates .

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(B) Meta plots showing H2A.Z occupancy on H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. RPKM, reads per kilobase per million. The H2A.Z ChIP-seq results were generated from two independent biological replicates.


( D ) Heat maps showing H2A . Z enrichment in WT , pie1 , chr11 / 17 , mbd9 - 1 mutants over PIE1

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(D) Heat maps showing H2A.Z enrichment in the WT, pie1, chr11/17, and mbd9-1 mutants over the PIE1


( D ) Heat maps showing H2A . Z enrichment in WT , pie1 , chr11 / 17 , mbd9 - 1 mutants over

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( E ) Shown are H2A . Z changes in pie1 , chr11 / 17 , mbd9 - 1 mutants relative to WT . PIE1 - dependent H2A . Z target genes were subjected to sorted by H2A . Z density in WT . color bar shows log2 ( fold change ) .

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(E) Shown are H2A.Z changes in the pie1, chr11/17, and mbd9-1 mutants relative to the WT. PIE1-dependent H2A.Z target genes were subjected to the analysis and sorted by H2A.Z density in the WT. The color bar shows log2(fold change).


( F ) Meta plots showing CHR11 PIE1 occupancy on PIE1 - dependent H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . CHR11 - Flag PIE1 - Flag ChIP - seq data are from two independent biological replicates .

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(F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates.


( G ) Heat maps showing enrichment of PIE1 - Flag CHR11 - Flag over PIE1 - dependent H2A . Z target genes . peaks were sorted by H2A . Z density in WT .

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(G) Heat maps showing the enrichment of PIE1-Flag and CHR11-Flag over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT.


( H ) Box plot showing enrichment of CHR11 PIE1 over H2A . Z target genes non - H2A . Z target genes . log2 ( fold change ) values of RPKM between CHR11 - Flag or PIE1 - Flag signals WT signals are shown . Center lines box edges represent medians interquartile range ( IQR ) , respectively . Whiskers extend to minimum maximum values within 1 . 5 times of IQR . data shown are from two independent biological replicates . difference between CHR11 - Flag PIE1 - Flag ChIP - seq was determined by student ' s t . * * P < 0 . 01 .

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(H) Box plot showing the enrichment of CHR11 and PIE1 over H2A.Z target genes and non-H2A.Z target genes. The log2(fold change) values of RPKM between the CHR11-Flag or PIE1-Flag signals and the WT signals are shown. Center lines and box edges represent medians and the interquartile range (IQR), respectively. Whiskers extend to minimum and maximum values within 1.5 times of the IQR. The data shown are from two independent biological replicates. The difference between the CHR11-Flag and PIE1-Flag ChIP-seq levels was determined by student's t test. ** P<0.01.


( A ) Increasing amounts of CHR11 ( left panel ) or PIE1 ( right panel ) were incubated with end - positioned ( lane 1 - 4 9 - 12 ) or center - positioned ( lane 5 - 8 13 - 16 ) mono - nucleosomes in presence of ATP . sliding results were then visualized on native PAGE gels by EB .

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(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.


( B ) Distribution of nucleosomes over TSS of - coding genes in WT , chr11 / 17 , pie1 , mbd9 - 1 mutants . MNase - seq results were generated from two independent biological replicates .

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(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.


( E ) Representative immunoblot for key lipolytic enzymes HSL ATGL expression ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . E , Unpaired Student ' s t - .

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(E) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5-6/group). Data are represented as mean ± s.e.m. E, Unpaired Student's t-test.


( F ) Immunoblot of autophagic flux in mWAT ( upper panel ) gWAT ( lower panel ) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 20mM NH4Cl for 4 hours or DMSO ( Vehicle ) ( n = 3 - 4 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

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(F) Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle) (n = 3-4/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( G ) Representative transmission electron microscopy from mesenteric adipose tissue 7 days post DSS - induced colitis induction . Lower panel is showing magnification of selected . arrows show autophagosomal structures .

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(G) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS-induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures.


( J ) Representative immunoblot for LC3 - I / - II ACTIN expression of autophagic flux in creeping fat tissues ( CrF ) adjacent mesenteric adipose tissues ( Ad . MAT ) of Crohn ' s disease ( n = 3 / group ) . Additionally , autophagic flux was determined in mesenteric adipose tissue ( MAT ) of a colorectal cancer as control ( dotted ) . Data are represented as mean ± s . e . m . ( J ) Paired Student ' s t - .

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(J) Representative immunoblot for LC3-I/-II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line). Data are represented as mean ± s.e.m. (J) Paired Student's t-test.


( C ) Representative immunoblot for LC3 - I LC3 - II expression of LC3 conversion ( LC3 - II / LC3 - I ) ( n = 3 / group ) . Data are represented as mean ± s . e . m . C , Unpaired Student ' s t - .

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(C) Representative immunoblot for LC3-I and LC3-II protein expression and quantification of LC3 conversion ratio (LC3-II/LC3-I) (n = 3/group). Data are represented as mean ± s.e.m. C, Unpaired Student's t-test.


( F ) Colon after two post - deletion ( steady state ; n = 14 / group ) after DSS at day 7 ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . , F , Unpaired Student ' s t - .

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(F) Colon length after two weeks post-deletion (steady state; n = 14/group) and after DSS at day 7 (n = 18-22/group). Data are represented as mean ± s.e.m. , F, Unpaired Student's t-test.


( G ) Representative H & E ( 10x magnification ) of colon sections of histological score at steady state ( n = 9 / group ) DSS colitis ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . G , Unpaired Student ' s t - .

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(G) Representative H&E staining images (10x magnification) of colon sections and quantification of histological score at steady state (n = 9/group) and DSS colitis (n = 18-22/group). Data are represented as mean ± s.e.m. G, Unpaired Student's t-test.


( I ) Absolute number CD45 + immune from colons at steady state ( n = 13 - 14 / group ) or at 7 days post - DSS induction ( n = 18 - 22 / group ) . ( J ) Frequency of myeloid population in colon at day 7 post - DSS induction ( n = 18 - 25 / group ) . ( K ) Absolute number of Ly6C + monocytes discriminated by absence or presence of MHCII for infiltrating inflammatory monocytes respectively ( n = 18 - 25 / group ) . Data are represented as mean ± s . e . m . I ) Unpaired Student ' s t - . ( J , K ) Two - Way ANOVA .

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(I) Absolute number CD45+ immune cells from colons at steady state (n = 13-14/group) or at 7 days post-DSS induction (n = 18-22/group). (J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 18-25/group). (K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 18-25/group). Data are represented as mean ± s.e.m. I) Unpaired Student's t-test. (J, K) Two-Way ANOVA.


( A ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 - 5 / group ) glycerol ( right , n = 7 - 8 / group ) in culture supernatant of adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h . ( B ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 / group ) glycerol ( right , n = 7 / group ) adipose tissue explants simulated with TNFα ( 100ng / mL ) for 24h before replacing with fresh medium in absence of TNFα for 3h . Data are represented as mean ± s . e . m . ( A , B , Two - Way ANOVA . ( B , Unpaired Student ' s t - .

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(A) Ex vivo lipolysis measured by released free fatty acid (left, n = 4-5/group) and glycerol (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol (10µM) for 1-2h. (B) Ex vivo lipolysis measured by released free fatty acid (left, n = 4/group) and glycerol (right, n = 7/group) adipose tissue explants simulated with TNFα (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα for 3h. Data are represented as mean ± s.e.m. (A, B, Two-Way ANOVA. (B, Unpaired Student's t-test.


( E ) of FFA species in serum in water - treated DSS - treated mice as measured by FID - GC ( n = 12 - 14 / group ) . Data are represented as mean ± s . e . m . E ) Two - Way ANOVA .

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(E) Concentration of individual FFA species in serum in water-treated and DSS-treated mice as measured by FID-GC (n = 12-14/group). Data are represented as mean ± s.e.m. E) Two-Way ANOVA.


( C ) Ex vivo lipolysis assays on Atg7 - deficient adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . ( C , Two - Way ANOVA .

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(C) Ex vivo lipolysis assays on Atg7-deficient adipose tissue explants simulated with isoproterenol (10µM) for 1-2h (n = 5-6/group). Data are represented as mean ± s.e.m. (C, Two-Way ANOVA.


( H ) Expression of pro - inflammatory cytokines in colon tissues at 7 days post - DSS induction ( n = 8 / group ) . Dotted represents non - inflamed controls . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

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(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction (n = 8/group). Dotted line represents non-inflamed controls. Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( K ) Absolute number of Ly6C + monocytes discriminated by absence or presence of MHCII for infiltrating inflammatory monocytes respectively ( n = 8 / group ) . Data are represented as mean ± s . e . m . K ) Unpaired Student ' s t - .

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(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 8/group). Data are represented as mean ± s.e.m. K) Unpaired Student's t-test.


( E ) Fold change expression of NRF2 - target genes in visceral adipocytes at day 7 after DSS induction from normalized of RNAseq dataset ( n = 6 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

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(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( H ) Representative immunoblot of EPHX1 EPHX2 in gonadal adipose tissues at day 7 after DSS induction . Asterix indicating non - specific band ( n = 14 - 18 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

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(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( B ) Colitis was induced in mice for 7 days adipose tissues were extracted cultured for 6 hours in serum - starved medium . Secretion of IL - 10 from mesenteric ( left panel ) gonadal adipose tissues ( right panel ) was measured by ELISA . Shapes identify experiments ( n = 5 - 15 / group ) . Data are represented as mean ± s . e . m . ( B ) Two - Way ANOVA with regression for experiment .

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(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group). Data are represented as mean ± s.e.m. (B) Two-Way ANOVA with regression for experiment.


( F ) Serum cytokines upon DSS - induced colitis at day 7 post - induction ( n = 17 - 23 / group ) . Data are represented as mean ± s . e . m . Two - Way ANOVA .

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(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.


( A ) of Il10 transcript in RAW264 . 7 upon stimulation to epoxy fatty ( n = 3 / group ) . ( B ) of IL - 10 in supernatant of RAW264 . 7 upon stimulation to epoxy fatty ( n = 3 / group ) . Data are represented as mean ± s . e . m . ( A , B ) One - Way ANOVA .

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(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). (B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA.


( C - G ) ssDNA repair efficiency of indicated TS mutants as determined from maximal final percentages of with Rad52 foci during time course ( right panels ) . were synchronized in G1 released into S phase in presence of 0 . 033 % MMS for 1 hour ( 60 + ) , treated with 2 . 5 % sodium thiosulfate to inactivate MMS , washed released into fresh medium for different times . They were also released into medium without MMS for 1 hour to control formation of spontaneous Rad52 - YFP foci ( 60 - ) . of with foci at each point was normalized to highest value of wild type , taken as 100 ( left panels ) . cycle progression was determined by sorting . mean of 3 ( wild type , rad18 ∆ , mms2 ∆ , rad51 ∆ , rad57 ∆ , rad54 ∆ ) , 4 ( pol30 - K164R ) 5 ( rad5 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - Student ' s t - are shown , where three asterisks represent p - values < 0 . 001 .

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

(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.


( C - E ) ssDNA repair efficiency of indicated TLS mutants as determined from maximal final percentages of with Rad52 foci during time course ( D E , right panel ) . were synchronized in G1 released into S phase in presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate MMS , washed released into fresh medium for different times . of with foci at each point was normalized to highest value of wild type , taken as 100 ( C E , left panel ) . Data information : mean of 3 - 12 12 ( wild type ) , 6 ( rev1 ∆ , rev3 ∆ , rad30 ∆ , rev1 ∆ rad30 ∆ , rev3 ∆ rad30 ∆ ) , 3 ( rev1 ∆ rev3 ∆ rev1 ∆ rev3 ∆ rad30 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - Student ' s t - are shown , where one , two three asterisks represent p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O 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-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( C - D ) ssDNA repair efficiency of rad5 ∆ rev1 ∆ mutants as determined from maximal final percentages of with Rad54 foci during time course ( right panels ) . were synchronized in G1 released into S phase in presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate MMS , washed released into fresh medium for different times . of with foci at each point was normalized to highest value of wild type , taken as 100 ( left panels ) . Data information : mean of independent experiments are shown . Statistically significant differences according to an unpaired two - Student ' s t - are shown , where one , two three asterisks represent p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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 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

(C-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


signal of RPA foci per ( E ) at times 1 or 2 ( peak ) 7 hours from time course shown in ( B ) . Data information : mean of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - Student ' s t - . One , two three asterisks represent p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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

fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( F ) ssDNA repair efficiency of indicated mutants as determined by ChIP against Rfa1 - YFP at ARS305 replication origin . were synchronized in G1 released into S phase in presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate MMS , washed released into fresh medium . amount of Rfa1 at ssDNA was determined at 1 7 hours after MMS release . RPA enrichment was calculated as between immunoprecipitate input values . values were normalized to wild type at 7 hours , taken as 1 . Data information : mean of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - Student ' s t - . One , two three asterisks represent p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively . In ( F ) values were significant relative to untagged controls ( not shown for clarity ) .

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(F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).


Effect of lack of HR factors in spontaneous MMS - induced mutagenesis . frequency of spontaneous MMS - induced forward mutagenesis at CAN1 locus was determined in mid - log phase cultures before ( spontaneous mutagenesis ) after treatment with 0 . 01 % MMS for 4 hours ( damage - induced ) . Thus , frequency of damage - induced mutagenesis includes mutants that arose spontaneously before MMS addition . frequency of mutagenesis was determined at 0 . 01 % MMS because at higher MMS concentrations loss of viability of rad mutants did not allow us to select for canavanine - resistant . Survival ( % ) after 4 hours in MMS relative to wild type ( taken as 100 ) is shown below genotypes . mean of 6 ( wild type ) , 9 ( rad54 ∆ ) 3 ( ) independent fluctuation are shown . Asterisks indicate statistically significant differences according to an unpaired two - Student ' s t - ( one , two three asterisks represent p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively ) . Only statistical of effect of rad52 ∆ , rad57 ∆ rad51 ∆ on rad54 ∆ - mediated mutagenesis is included for clarity . mutants displayed statistically significant differences with wild type not with each other in absence of MMS .

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Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.


( A ) Effect of rad54 ∆ rad52 ∆ mutations in spontaneous UV - induced uSCE . frequency of spontaneous UV - induced recombinants was determined by irradiating or not plated onto solid medium after corresponding dilutions ( SMM SMM without histidine to calculate total recombinant , respectively ) . mean of 3 - 5 independent fluctuation are shown . Asterisks indicate statistically significant differences according to an unpaired two - Student ' s t - . Two three asterisks represent p - values < 0 . 01 < 0 . 001 , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.


( B ) Rad6 binds to in absence presence of MMS . ChEC of G1 - synchronyzed released in absence or presence of 0 . 033 % MMS for indicated times . Data information : gel electrophoresis , content ( sorting ) of digestion profiles are shown . experiments were repeated twice with similar results .

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(B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( C - F ) Rad6 binding to depends on Rad18 , Rad52 ( C ) , Rad51 ( D ) , Rad57 ( E ) but not Rad54 ( F ) as determined by ChEC analyses of asynchronous cultures without or with 0 . 05 % MMS for 2 hours . Data information : gel electrophoresis , content ( sorting ) of digestion profiles are shown . experiments were repeated twice with similar results .

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

(C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( A - D ) Effect of rad52 ∆ , rad51 ∆ ( A ) , rad57 ∆ ( B ) rad54 ∆ ( C ) on PCNA ubiquitylation in synchronized in G1 released into fresh medium in presence of 0 . 025 % MMS . cycle progression was followed by sorting western blot against Clb2 . Pgk1 was used as loading control . amounts of mono - polyubiquitylated PCNA were normalized to total amount of PCNA . For each time point , rad mutant values were relativized to those of wild type , taken as 1 . mean of 3 independent experiments are shown ( D ) . An asterisk indicates a mean significantly different than 1 , according to a One - sample t - .

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(A-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.


( A ) Immunofluorescence against Iba - 1 in cortex ( Cx ) , hippocampus ( Hip ) cerebellum ( Cb ) of NPA affected control 3 year - old children . Scale bars , 50 μm . ( B ) Magnified image showing ramified ( left ) versus amoeboid ( right ) in control NPA microglia . Scale bars , 10 μm . ( C ) Mean ± of Iba - 1 positive in different brain regions from a NPA affected a control child ( n = 20 , Student ´ s t - ) .

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(A) Immunofluorescence staining against Iba-1 in cortex (Cx), hippocampus (Hip) and cerebellum (Cb) of NPA affected and control 3 year-old children. Scale bars, 50 μm. (B) Magnified image showing ramified (left) versus amoeboid (right) morphology in control and NPA microglia. Scale bars, 10 μm. (C) Mean ± SEM area of Iba-1 positive cells in different brain regions from a NPA affected and a control child (n = 20 cells, Student´s t-test).


( D ) Immunofluorescence against Iba - 1 in Cx , Hip Cb of 3 month - old ASMko wt mice . DAPI shows nuclei . Scale bars , 50 μm .

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(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.


( E ) Mean ± number of Iba - 1 positive in different brain regions from ASMko wt mice ( n = 5 mice per group , Student ´ s t - ) .

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(E) Mean ± SEM number of Iba-1 positive cells in different brain regions from ASMko and wt mice (n = 5 mice per group, Student´s t-test).


( F ) 3D rendered obtained with Imaris Surface software from high magnification of immunofluorescence against Iba - 1 showing representative microglial in Cx , Hip Cb of ASMko wt mice . Scale bars , 10 μm .

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(F) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of ASMko and wt mice. Scale bars, 10 μm.


( G ) Mean ± of Iba - 1 positive in different brain regions from ASMko wt mice ( n = 5 mice per group , > 10 per mouse , Student ´ s t - ) . Scale bars , 15 μm .

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(G) Mean ± SEM area of Iba-1 positive cells in the different brain regions from ASMko and wt mice (n = 5 mice per group, >10 cells per mouse, Student´s t-test). Scale bars, 15 μm.


( H ) Example of projection detection by Filament Imaris software . ( I ) Mean ± branch point number per process process in iba1 + obtained with Imaris Filament software ( Student ´ s t - ) .

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(H) Example of projection detection by the Filament Imaris software. (I) Mean ± SEM branch point number per process and process length in iba1+ cells obtained with the Imaris Filament software (Student´s t-test).


( A ) Immunofluorescence against Iba - 1 in Cb of ASMko wt mice treated or not with PLX for 2 months . DAPI shows nuclei . Scale bar , 100 μm . ( B ) Magnified ( from A ) showing ramified ( left ) versus amoeboid ( right ) in wt ASMko microglia treated or not with PLX . Scale bars , 30 μm . ( C ) Mean ± number of Iba - 1 positive in Cb of different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm. (B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm. (C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( D , E ) Performance in Rotarod of ASMko wt mice treated or not with PLX for 2 months expressed as mean ± time spent in rotating rod on each of four trials ( D ) or mean ± time of four trials ( E ) ( n = 7 mice per group , Two - way Anova , Games - Howell ( D ) Bonferroni ( E ) post hoc ) .

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(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).


( F ) Mean ± of ASMko wt mice treated or not with PLX at indicated time during long - term treatment ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).


( G ) Survival curve of ASMko wt mice with or without long - term PLX treatment ( n = 7 mice per group ) .

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(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).


( H ) Immunofluorescence against PC marker Calbindin in mid posterior lobules of Cb in ASMko wt mice treated or not with PLX for 2 months . DAPI shows nuclei . Scale bar , 500 μm . ( I ) Mean ± number of Calbindin positive in anterior ( lobules I - V ) , middle ( lobules VI - VIII ) posterior zone ( lobules IX - X ) of Cb from different mouse groups ( n = 7 mouse per group , Two - way Anova , Bonferroni post hoc ) .

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(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm. (I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mouse per group, Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence against CD16 / CD32 or Arg - 1 in Cb of ASMko mice ( red ) . Microglia were identified by F4 / 80 or Iba - 1 ( green ) . DAPI shows nuclei . Scale bar , 50 μm . ( B ) Mean ± number of Arg - 1 CD16 / CD32 positive microglia per in Cb from ASMko wt mice ( n = 6 mice per group ) .

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(A) Immunofluorescence staining against CD16/CD32 or Arg-1 in Cb of ASMko mice (red). Microglia were identified by F4/80 or Iba-1 staining (green). DAPI staining shows cell nuclei. Scale bar, 50 μm. (B) Mean ± SEM number of Arg-1 and CD16/CD32 positive microglia per area in Cb from ASMko and wt mice (n = 6 mice per group).


( C ) Mean ± of TNFa in Cb from ASMko wt mice measured by ELISA ( n = 7 mice per group , Student ´ s t - ) .

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(C) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA (n = 7 mice per group, Student´s t-test).


( D ) Mean ± of TNFa mRNA in Cb of ASMko wt mice determined by qRT - PCR ( n = 7 mice per group , Student ´ s t - ) .

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(D) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT-PCR (n = 7 mice per group, Student´s t-test).


( E ) Survival after LPS challenge in ASMko wt mice treated or not with dexamethasone ( n = 8 mice per group ) .

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(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).


( F ) Immunofluorescence against MBP in Cb of ASMko wt mice treated or not with PLX for 2 months . Scale bar , 50 μm .

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(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.


( G ) Mean ± MBP in different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(G) Mean ± SEM MBP intensity in the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( H ) High magnification of immunofluorescence against MBP in ASMko mice treated or not with PLX for 2 months . arrows show MBP indicative of myelin debris . Scale bar , 50 μm . ( I ) Mean ± number of MBP per in different mouse groups ( n = 7 mice per group , Student ´ s t - ) .

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(H) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months. White arrows show MBP aggregates indicative of myelin debris. Scale bar, 50 μm. (I) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups (n = 7 mice per group, Student´s t-test).


( J ) High magnification their 3D rendered replicates from cerebellar wt ASMko microglia identified by Iba - 1 of myelin marker MBP . Scale bars , 10 μm .

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(J) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba-1 and of the myelin marker MBP. Scale bars, 10 μm.


( K ) Amplified image its 3D rendered replicate of amoeboid microglia containing myelin debris ( arrowheads ) in ASMko cerebellum . orthogonal projection of this in lower panels confirms presence of myelin debris inside underneath plasma ( arrowhead ) . DAPI shows nuclei . Scale bars , 10 μm . ( L ) Mean ± of microglia showing internalised myelin debris in wt ASMko mice ( n = 7 mice per group ) .

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(K) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris (arrowheads) in the ASMko cerebellum. The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane (arrowhead). DAPI staining shows cell nuclei. Scale bars, 10 μm. (L) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice (n = 7 mice per group).


( A ) Transmission electron microscopy image of microglia in Cb of ASMko mice showing accumulation of multillamelar vacuoles other lysosome related vesicles filling whole cytoplasm of these . Arrowhead identifies nuclei . Scale bar , 5 μm . On right , higher magnification showing a multillamelar ( arrowhead ) with characteristic concentric lamellar inclusions . Scale bar , 0 . 5 μm .

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(A) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells. Arrowhead identifies the nuclei. Scale bar, 5 μm. On the right, higher magnification showing a multillamelar body (arrowhead) with the characteristic concentric lamellar inclusions. Scale bar, 0.5 μm.


Immunofluorescence against Galectin3 - GFP in BV2 microglial treated with or without SM ( B ) Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) Scale bar, 10 μm.


Immunofluorescence against Galectin3 - GFP in BV2 microglial treated with or without Siramesine ( C ) . Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without Siramesine (C). Scale bar, 10 μm.


( D ) Mean ± number of Galectin3 - GFP positive vesicles in BV2 microglial in different conditions ( n = 4 independent cultures , > 50 per culture , Student ´ s t - ) .

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(D) Mean ± SEM number of Galectin3-GFP positive vesicles in BV2 microglial cells in the different conditions (n = 4 independent cultures, >50 cells per culture, Student´s t-test).


( E ) Lysotracker - Red in BMDMs from wt ASMko mice . Scale bar , 10 μm .

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(E) Lysotracker-Red staining in BMDMs from wt and ASMko mice. Scale bar, 10 μm.


( F ) Western blot of CathB GAPDH in fractions of wt ASMko BMDMs extracted with digitonin ( left ) . Mean ± pro - CathB CathB in fractions ( right ) ( n = 2 cultures per genotype , Student ´ s t - ) .

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(F) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin (left). Mean ± SEM pro-CathB and CathB levels in the cytosolic fractions (right) (n=2 cultures per genotype, Student´s t-test).


( G ) Immunofluorescence against Iba - 1 ( green ) CathB ( red ) in Cb of ASMko wt mice . Arrowheads show double positive . Scale bar , 50 μm . Below , magnified of selected areas . Scale bar , 20 μm . ( H ) Immunofluorescence against CathB ( green ) , Arg - 1 ( red , anti - inflammatory microglia ) F4 / 80 ( yellow , microglia ) in Cb of ASMko mice . DAPI shows nuclei . Scale bar , 50 μm . Below , magnified of selected areas . Scale bar , 20 μm . ( I ) Table showing mean ± of microglia presenting CathB ( CathB + / Iba - 1 + ) , number of CathB positive microglia co - expressing Arg - 1 ( CathB + / Arg - 1 + ) in Cb of ASMko wt mice ( n = 5 mice , > 10 per mouse ) .

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(G) Immunofluorescence staining against Iba-1 (green) and CathB (red) in Cb of ASMko and wt mice. Arrowheads show double positive cells. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (H) Immunofluorescence staining against CathB (green), Arg-1 (red, anti-inflammatory microglia) and F4/80 (yellow, all microglia) in Cb of ASMko mice. DAPI staining shows cell nuclei. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (I) Table showing mean ± SEM percentage of microglia presenting CathB staining (CathB +/Iba-1+), and the number of CathB positive microglia co-expressing Arg-1 (CathB+/Arg-1+) in Cb of ASMko and wt mice (n = 5 mice, >10 cells per mouse).


( A ) Western blot of CathB ( for both precursor ( pro - CathB ) - mature forms ) in culture media from ASMko wt BMDMs . of Ponceau - S is shown as loading control . ( B ) Mean ± fold increase in CathB in culture media from wt ASMko BMDMs ( n = 5 independent cultures , Student ´ s t - ) .

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(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control. (B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test).


( C ) Immunofluorescence against LAMP - 1 ( yellow ) Iba - 1 ( red ) in Cb of ASMko wt mice . Scale bar , 50 μm , zoomed 20 μm .

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(C) Immunofluorescence staining against LAMP-1 (yellow) and Iba-1 (red) in Cb of ASMko and wt mice. Scale bar, 50 μm, zoomed images 20 μm.


( D ) Immunofluorescence against LAMP - 1 in non - permeabilised BMDMs from ASMko wt mice . Scale bar , 50 μm .

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(D) Immunofluorescence staining against LAMP-1 in non-permeabilised BMDMs from ASMko and wt mice. Scale bar, 50 μm.


( E ) Mean ± of LAMP - 1 on surface of ASMko wt BMDMs ( n = 4 independent cultures , > 40 per culture , Student ´ s t - ) .

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(E) Mean ± SEM of LAMP-1 levels on the surface of ASMko and wt BMDMs (n = 4 independent cultures, >40 cells per culture, Student´s t-test).


( F ) Mean ± microvesicle exosome numbers in culture media from ASMko wt BMDMs ( n = 6 independent cultures , Student ´ s t - ) .

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(F) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs (n = 6 independent cultures, Student´s t-test).


( G ) Western blot of CathB Flotillin1 in secreted exosome fractions from ASMko wt BMDMs . ( H ) Western blot of CathB in exosome depleted culture media from wt ASMko BMDMs .

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(G) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs. (H) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs.


( I ) Mean ± MTT absorbance reflecting viability in neurons from wt mice treated with conditioned media from ASMko wt BMDMs in presence or absence of CathB inhibitor Ca074 ( n = 4 independent cultures Two - way Anova , Bonferroni post hoc ) .

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(I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc).


( J ) Immunofluorescence against Iba - 1 ( green ) CathB ( red ) in lobes of Cb sensitive or resistant to Purkinje . Asterisks indicate Purkinje in resistant lobes . Scale bar , 50 μm .

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(J) Immunofluorescence staining against Iba-1 (green) and CathB (red) in lobes of the Cb sensitive or resistant to Purkinje cell death. Asterisks indicate Purkinje cells in the resistant lobes. Scale bar, 50 μm.


( K ) Mean ± of CathB positive microglia in resistant sensitive Cb lobules of ASMko mice ( n = 5 mice , χ2 ) .

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(K) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice (n = 5 mice, χ2 test).


( L ) Western blot of Cath B β - Actin in cerebellar extracts of wt ASMko mice treated or not with PLX . ( M ) Mean ± of CathB normalized by β - Actin in cerebellar extracts of wt ASMko mice treated or not with PLX ( n = 6 mice Two - way Anova , Bonferroni post hoc ) .

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(L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX. (M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc).


( O ) Western blot of CathB ( for both precursor ( pro - CathB ) - mature forms ) in culture media from ASMko wt cerebellar organotypic cultures from mice fed with vehicle ( veh ) or PLX ( PLX ) . Below , Western blots of organotypic culture lysates against Iba - 1 , to confirm microglia depletion , against GAPDH as loading control .

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(O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control.


( P ) Mean ± increase in pro - CathB ( left ) CathB ( right ) in culture media from cerebellar organotypic cultures from wt ASMko mice treated or not with PLX ( n = 3 mice per group Two - way Anova , Bonferroni post hoc ) .

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(P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Mean ± of ASMko wt mice treated or not with Ca074Me at indicated time during treatment ( n = 7 mice per group ) .

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(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group).


Performance in Rotarod of ASMko wt mice treated or not with Ca074Me for 1 month expressed as mean ± time spent in rotating rod on each of four trials ( B )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B)


Performance in Rotarod of ASMko wt mice treated or not with Ca074Me for 1 month expressed as mean ± time of four trials ( C )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials (C)


( D ) Immunofluorescence against PC marker Calbindin in posterior lobules of Cb in ASMko wt mice treated or not with Ca074Me for 1 month . DAPI shows nuclei . Scale bar , 200 μm .

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(D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm.


( E ) Mean ± number of Calbindin positive in posterior zone ( lobules IX - X ) of Cb from different mouse groups ( n = 7 mice per group Two - way Anova , Bonferroni post hoc ) .

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(E) Mean ± SEM number of Calbindin positive cells in the posterior zone (lobules IX-X) of the Cb from the different mouse groups (n= 7 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence against Iba - 1 in Cx , Hip Cb of 2 month - old Npc1nmf164 wt mice . DAPI shows nuclei . Scale bars , 30 μm .

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(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm.


( B ) 3D rendered obtained with Imaris Surface software from high magnification z - stack of immunofluorescence against Iba - 1 showing representative microglial in Cx , Hip Cb of Npc1nmf164 wt mice . Scale bars , 10 μm .

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(B) 3D rendered images obtained with Imaris Surface software from high magnification z-stack images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of Npc1nmf164 and wt mice. Scale bars, 10 μm.