D . Representative of P5 mouse seminiferous tubule 10 µm cryo - sections stained for PLZF ( red ) , ODF2 ( green ) , NIN ( magenta ) PH3 ( blue ) , focusing on PLZF + germ ( GSCs ) in prometaphase . Scale bars : 2 μm . Yellow lines highlight basement . Yellow arrowheads highlight mother centrosomes arrowheads highlight daughter centrosomes .

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D. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue), focusing on PLZF+ germ stem cells (GSCs) in prometaphase. Scale bars: 2 μm. Yellow lines highlight basement membrane. Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes.


E . of of prometaphase GSCs which preferentially position their mother centrosome close to basement in CEP250 + / + CEP250 - / - testis at P5 . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 50 were analyzed in each mouse average was calculated for each mouse . Data information : In ( E ) , data are presented as mean ± . * * * * P < 0 . 001 ( unpaired Student ' s t - ) .

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E. Quantification of Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250+/+ and CEP250-/- testis at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In (E), data are presented as mean ± SEM. ****P < 0.001 (the unpaired Student's t-test).


( a ) Confocal microscopy of synchronized HeLa stained for VHR ( green ) BrdU ( red ) to indicate S - phase entry , ( DAPI ; blue ) . were synchronized using a double thymidine block then washed placed in culture for indicated times . shown are representative of majority of in each sample .

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(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample.


b ) Anti - VHR , anti - actin anti - Prc1immunoblots of lysates of same .

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b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells.


( c ) histograms of same at indicated time points .

(c) DNA histograms of the same cells at the indicated time points.


( d ) Confocal microscopy of HeLa at various stages of mitosis stained for tubulin ( green ) , VHR ( red ) ( DAPI ; blue ) .

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(d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue).


( e ) Anti - VHR immunoblot of synchronized from G1 , S , G2 M phase treated for 30 min with 50 μg ml − 1 of synthesis inhibitor cycloheximide . Note that only in G1 showed a rapid loss of VHR . scale bars represent 5 μm in a 2 μm in b .

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(e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.


( a ) Anti - VHR anti - actin immunoblots of lysates of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for indicated number of days .

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(a) Anti-VHR and anti-actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days.


( b ) Number of at start of experiment ( 200 , 000 ) on day 3 for HeLa transfected with a control luciferase siRNA ( black bars ) or with VHR siRNA ( bars ) .

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(b) Number of cells at the start of the experiment (200,000) and on day 3 for HeLa cells transfected with a control luciferase siRNA (black bars) or with VHR siRNA (white bars).


( c ) microscopy of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for three days .

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(c) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days.


( d ) Thymidine incorporation by HeLa transfected with a control luciferase siRNA or with VHR siRNA . data represent mean ± sd . ( n = 3 ) .

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(d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3).


( e ) β - galactosidase of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for three days . Note increased number of blue - granules in many . (

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(e) β-galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. Note the increased number of blue-staining granules in many cells. (


( f ) Telomerase activity of treated with indicated concentrations of VHR siRNA . Each sample was analysed in triplicate , with RNAse added to a fourth sample .

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(f) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA. Each sample was analysed in triplicate, with RNAse added to a fourth sample.


( g ) Anti - VHR immunoblot of same as in f . ( h ) Anti - actin control immunoblot of same as in f . scale bars represent 10 μm in c 25 μm in e .

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(g) Anti-VHR immunoblot of the same cells as in f. (h) Anti-actin control immunoblot of the same cells as in f. The scale bars represent 10 μm in c and 25 μm in e.


( a ) - cycle distribution number of transfected with control or VHR siRNA observed for 4 - 5 days . were stained with propidium iodide for content analysed by FACS after indicated number of days in culture .

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(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture.


( b ) Confocal microscopy of same HeLa stained for VHR ( green ) BrdU ( red ) to indicate S - phase entry , ( DAPI , blue ) . Control siRNA - treated treated with VHR are shown . bottom panels represent merges of 3

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(b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above


( c ) of BrdU positive in b . indicated numbers of were counted data are given as of BrdU positive of this total number .

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(c) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number.


d ) of for phosphoH3 . indicated number of were counted data are given as of phosphoH3 - positive of this total number .

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d) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number.


( e ) Confocal microscopy of control or VHR siRNA - transfected HeLa stained for VHR ( green ) phospho H3 ( red ) to indicate M - phase entry , ( DAPI , blue ) . bottom panels represent merges of above .

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(e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above.


( f ) Immunoblots of total lysates of control siRNA transfected VHR siRNA with indicated antibodies . Note that VHR was downregulated by approximately 80 % , wheras actin were unchanged .

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(f) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged.


( g ) Immunoblot with anti - p21Cip1 - WAF1 of total lysates of transfected with control siRNA ( lanes 1 - 2 ) , two different VHR siRNAs ( lanes 3 - 6 ) or another control siRNA ( lanes 7 - 8 ) . scale bars represent 10 μm in b e .

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(g) Immunoblot with anti-p21Cip1-WAF1 of total lysates of cells transfected with control siRNA (lanes 1-2), two different VHR siRNAs cells (lanes 3-6) or another control siRNA (lanes 7-8). The scale bars represent 10 μm in b and e.


( a ) Anti - VHR immunoblots of same lysates used for assessment of MAP kinase activation in b - e . were stimulated with 10 % serum for 0 , 1 , 5 , 15 , 30 60 min , as indicated at bottom of figure . Equal amounts of total were loaded into each lane . ( b - e ) Immunoblots of same lysates with indicated antibodies for assement of MAP kinase activation .

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(a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b-e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b-e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.


( a ) Proliferation of transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek , Jnk or Mek plus Jnk siRNAs . data are calculated as of control ( control siRNA alone ) represent mean ± s . d . from three independent experiments . starting numbers were 150 , 000 or 200 , 000 control had increased by 5 . 3 , 5 . 0 13 - fold in three experiments .

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(a) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments.


( b ) Immunoblots of same with indicated antibodies .

(b) Immunoblots of the same cells with the indicated antibodies.


A . Live - imaging to detect mitotic perturbations . HeLa stably expressing a marker ( H2B - mCherry ; red ) a import substrate ( IBB - eGFP ; green ) were imaged by automated live - microscopy duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing on time from envelope breakdown ( mitotic entry ) until reassembly ( mitotic exit ) ( Schmitz et al . , 2010 ) .

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A. Live-cell imaging assay to detect mitotic perturbations. HeLa cells stably expressing a chromatin marker (H2B-mCherry; red) and a nuclear import substrate (IBB-eGFP; green) were imaged by automated live-cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown (mitotic entry) until nuclear reassembly (mitotic exit) (Schmitz et al., 2010).


B . miRNA mimic screen for miRNAs that regulate mitosis genes . miRNA - mimics from a library of embryonic cortically expressed miRNAs were transfected individually into HeLa prometaphase to anaphase onset duration was determined as in A . points correspond to mean z - score of duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA - mimic .

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B. miRNA mimic screen for miRNAs that regulate mitosis genes. miRNA-mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A. Individual points correspond to the mean z-score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA-mimic.


C . Cumulative histograms of mitotic progression for control for transfected with miRNA mimics . envelope breakdown is at t = 0 min . ( n ≥ 71 in conditions ) .

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C. Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics. Nuclear envelope breakdown is at t = 0 min. (n ≥ 71 in all conditions).


D . Confocal time lapse microscopy of HeLa stably expressing H2B - mCherry ( red ) a microtubule marker ( α - Tub - eGFP ; green ) 48 h after transfection of miR - 449a mimic , or non - targeting control siRNA . Scale bar , 10 μm . E . of spindle rotation duration from prometaphase to anaphase onset in time - lapse movies as in D . miR - 449a mimic promotes spindle rotation . Duration from prometaphase to anaphase onset was defined as time from envelope breakdown to anaphase onset . Spindle rotation during metaphase was measured as described in methods . data points correspond to single ( n ≥ 72 in conditions ) . Normality was tested with Kolmogorov - Smirnov . Variance between samples was tested using F . Significance was tested by Welch ' s t : p - value = 2 . 598e - 06 comparing spindle rotation of negative control versus miR - 449a mimic - transfected ( data points are compared ) . p - value = 0 . 0005621 ( for with prometaphase - anaphase onset duration lower than 60 min .

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D. Confocal time lapse microscopy images of HeLa cells stably expressing H2B-mCherry (red) and a microtubule marker (α-Tub-eGFP; green) 48 h after transfection of miR-449a mimic, or non-targeting control siRNA. Scale bar, 10 μm.E. Quantification of spindle rotation and duration from prometaphase to anaphase onset in time-lapse movies as in D. miR-449a mimic promotes spindle rotation. Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset. Spindle rotation during metaphase was measured as described in methods. Individual data points correspond to single cells (n ≥ 72 in all conditions). Normality was tested with Kolmogorov-Smirnov test. Variance between samples was tested using F test. Significance was tested by Welch's t test: p-value = 2.598e-06 comparing spindle rotation of negative control versus miR-449a mimic-transfected cells (all data points are compared). p-value = 0.0005621 (for cells with prometaphase-anaphase onset duration lower than 60 min.


A . expression of endogenous miR - 34 / 449 family members were measured by RT - qPCR in ventricular zone samples derived by microdissection of mouse cortices at E14 . of different miR - 34 / 449 family members miR - 7a - 1 , a highly expressed miRNA relevant in cortical progenitor biology , were determined . concentrations were normalized ( norm . ) using miR - 7a - 1 ( n = 8 cortices , 2 different litters ) . Error bars indicate standard error .

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A. The expression levels of endogenous miR-34/449 family members were measured by RT-qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14. The levels of the different miR-34/449 family members and miR-7a-1, a highly expressed miRNA relevant in cortical progenitor biology, were determined. All concentrations were normalized (norm.) using miR-7a-1 concentration (n = 8 cortices, 2 different litters). Error bars indicate standard error.


B , C . Expression of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b miR - 34c are preferentially expressed in subventricular ( SVZ ) ventricular ( VZ ) zones of neocortex . Scale bar , 50 μm ( A ) , 10 μm ( B ) .

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B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (A), 10 μm (B).


B , C . Expression of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b miR - 34c are preferentially expressed in subventricular ( SVZ ) ventricular ( VZ ) zones of neocortex . Scale bar , 50 μm ( B ) , 10 μm ( C ) .

B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (B), 10 μm (C).


D , E . Brains of adult mice ( P23 ) , of brain . Dots indicate brains ; red indicates median . Mice lacking miR - 449abc miR - 34bc ( DKO ) or miR - 449abc , miR - 34bc , miR - 34a ( TKO ) have significantly smaller brains compared to littermate controls ( Het ) . Significance was tested by Pairwaise t - with Bonferroni correction ; Het ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00215 ; Het ( n = 6 brains , 2 different litters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00054 . Subfigure G was statistically tested as in E . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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D, E. Brains of adult mice (P23), and quantification of brain weight. Dots indicate individual brains; red line indicates median. Mice lacking miR-449abc and miR-34bc (DKO) or miR-449abc, miR-34bc, and miR-34a (TKO) have significantly smaller brains compared to littermate controls (Het). Significance was tested by Pairwaise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00215; Het (n = 6 brains, 2 different litters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00054. Subfigure G was statistically tested as in E. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


F , G . Confocal of coronal brain sections ( P23 ) of cortex width from miR - 34 / 449 DKO TKO mice littermate controls ( Het ) . Sections were stained with DAPI . DKO or TKO mice have significantly thinner cortices compared to littermate controls ( Het ) in adult mice ( P23 ) . ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00084 , Het ( n = 6 brains , 2 different liters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00211 . Scale bar , 500 μm .

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F, G. Confocal images of coronal brain sections (P23) and quantification of cortex width from miR-34/449 DKO and TKO mice and littermate controls (Het). Sections were stained with DAPI. DKO or TKO mice have significantly thinner cortices compared to littermate controls (Het) in adult mice (P23). (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00084, Het (n = 6 brains, 2 different liters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00211. Scale bar, 500 μm.


A . Confocal of coronal sections from E16 brains of miR - 34 / 449 KO mice littermate controls ( Het ) , stained with anti - Satb2 - antibody to label neurons of layers II - IV ( green ) DAPI to label nuclei ( blue ) . Scale bars : 50 μm . B . of number of Satb2 - positive per 100 μm ventricular zone surface . Bars indicate mean ± , as for quantifications shown in this figure ; n = 3 brains per genotype group , from two independent litters . Data was normalized ( norm . ) to ventricular zone surface analyzed ( 100 µm ) relativized to heterozygous control average value . Significance was tested by Welch ' s t : p - value = 0 . 01146 ( Het vs . KO ) ( n = 4 cortices per genotype group , 2 independent litters ) . subfigures were statistically tested as in B . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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A. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Satb2-antibody to label neurons of layers II-IV (green) and DAPI to label all cell nuclei (blue). Scale bars: 50 μm.B. Quantification of the number of Satb2-positive cells per 100 μm ventricular zone surface. Bars indicate mean ± SEM, as for all quantifications shown in this figure; n = 3 brains per genotype group, from two independent litters. Data was normalized (norm.) to the ventricular zone surface analyzed (100 µm) and relativized to the heterozygous control average value. Significance was tested by Welch's t test: p-value = 0.01146 (Het vs. KO) (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in B. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


C - J . Confocal density quantifications as in A using molecular markers for different types : C , D . E16 brains stained with anti - Ctip2 antibody to label layer Vneurons ( n = 4 brains per genotype group , 2 independent litters ) . Scale bars : 50 μm . in C were taken from same brain slices as shown in A . p - value = 0 . 03224 ( Het vs . KO ) ( immuno co - of Ctip2 ( red , C ) ) .

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C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:C, D. E16 brains stained with anti-Ctip2 antibody to label layer Vneurons (n = 4 brains per genotype group, 2 independent litters). Scale bars: 50 μm. Images in C were taken from same brain slices as shown in A. p-value = 0.03224 (Het vs. KO) (immuno co-staining of Ctip2(red, C)).


C - J . Confocal density quantifications as in A using molecular markers for different types : E , F . E14 brains stained with anti - Tbr1 antibody to label layer IVneurons p - value = 0 . 01392 ( Het vs . KO ) ( n = 3 4 brains per genotype group from 2 independent litters ) . Scale bars : 50 μm .

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C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:E, F. E14 brains stained with anti-Tbr1 antibody to label layer IVneurons p-value = 0.01392 (Het vs. KO) (n = 3 and 4 brains per genotype group from 2 independent litters). Scale bars: 50 μm.


C - J . Confocal density quantifications as in A using molecular markers for different types : G , H . E16 brains stained with anti - Tbr2 antibody to label intermediate progenitors . p - value = 0 . 01647 ( Het vs . KO ) ( n = 7 brains for each genotype group , 5 independent litters ) . Scale bars : 50 μm .

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C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:G, H. E16 brains stained with anti-Tbr2 antibody to label intermediate progenitors. p-value = 0.01647 (Het vs. KO) (n = 7 brains for each genotype group, 5 independent litters). Scale bars: 50 μm.


C - J . Confocal density quantifications as in A using molecular markers for different types : I , J . E16 brains stained with anti - Pax6 antibody to label radial glia progenitors . p - value = 0 . 002768 ( n = 5 brains per genotype group , 3 independent litters ) . Scale bars : 50 μm .

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C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:I, J. E16 brains stained with anti-Pax6 antibody to label radial glia progenitors. p-value = 0.002768 (n = 5 brains per genotype group, 3 independent litters). Scale bars: 50 μm.


K . 3D reconstruction of a dividing radial glial progenitor at early anaphase . A coronal section of an E14 brain of a heterozygous control mouse was stained with anti - phospho - Vimentin antibody ( red ) , anti - γ - tubulin antibody ( green / yellow ) , phalloidin ( magenta ) , DAPI ( blue ) imaged by 3D confocal microscopy . " s " indicates spindle axis , " α " indicates angle relative to ventricular surface plane , which was determined by a vector path as indicated by thin lines located on left side of image . Yellow dots highlight centrosomes of spindle poles from analyzed dividing . Scale bars : 5 μm . L . of spindle orientation in radial glial as in K for E14 brains of miR - 34 / 449 KO littermate controls ( Het ) . Each dot represents a single dividing ; p - value = 3 . 166e - 05 ( Het vs . KO ) ( n = 131 vs . 107 , n = 4 brains per genotype group , 2 independent litters ) .

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K. 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti-phospho-Vimentin antibody (red), anti-γ-tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. "s" indicates spindle axis, "α" indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Scale bars: 5 μm.L. Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR-34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; p-value = 3.166e-05 (Het vs. KO) (n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters).


A . of mRNA expression by RT - qPCR of candidate miR - 34 / 449 targets that might regulate spindle orientation in E14 cortex samples from miR - 34 / 449 KO mice compared with littermate controls ( Het ) . Expression was normlaized ( norm . ) to Phosphoglycerate kinase ( PGK ) mRNA . Significance was tested by Welch ' s t : p = 0 . 009756 ( JAM - A ( Het ) vs . JAM - A ( KO ) ) , Het vs . KO comparisons p = n . s . ( n = 4 cortices per genotype group , 2 independent litters ) . subfigures were statistically tested as in A . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Red bar indicates median .

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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O 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. Quantification of mRNA expression by RT-qPCR of candidate miR-34/449 targets that might regulate spindle orientation in E14 cortex samples from miR-34/449 KO mice compared with littermate controls (Het). Expression was normlaized (norm.) to Phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch's t test: p = 0.009756 (JAM-A (Het) vs. JAM-A (KO)), all the rest Het vs. KO comparisons p=n.s. (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in A. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. Red bar indicates median.


B . RT - qPCR of JAM - A mRNA expression in HeLa 48 h after transfection of miR - 449a mimic compared to transfected with non - targeting negative control siRNA . JAM - A mRNA Expression was normalized ( norm . ) to Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) mRNA . p = 0 . 0001218 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 measurements from 3 independent replicates ) .

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

B. RT-qPCR quantification of JAM-A mRNA expression in HeLa cells 48 h after transfection of miR-449a mimic compared to cells transfected with non-targeting negative control siRNA. JAM-A mRNA Expression was normalized (norm.) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. p = 0.0001218 (Neg. ctrol. vs. miR-449a mimic) (n = 9 measurements from 3 independent replicates).


C , D . mouse J110 were transfected with either miR - 449a mimic or a negative control mimic . Whole extracts were subjected to immunoblot to detect endogenous JAM - A ACTIN . ACTIN was used to normalize JAM - A expression ( norm . ) as loading control . p = 0 . 04454 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 4 independent wells , 2 independent transfections ) .

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

C, D. mouse J110 cells were transfected with either miR-449a mimic or a negative control mimic. Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM-A and ACTIN. ACTIN was used to normalize JAM-A expression (norm.) as loading control. p = 0.04454 (Neg. ctrol. vs. miR-449a mimic) (n = 4 independent wells, 2 independent transfections).


E , F . HeLa were cotransfected with either miR - 449a mimic or a negative control mimic , together with JAM - A - GFP fusion GFP . Whole extracts were subjected to immunoblot to detect GFP . GFP was used to normalize ( norm . ) by transfection efficiency as loading control . p = 0 . 001176 ( JAM - A WT Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 independent wells , 4 independent transfections ) .

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 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 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

E, F. HeLa cells were cotransfected with either miR-449a mimic or a negative control mimic, together with JAM-A-GFP fusion protein and GFP. Whole cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. p = 0.001176 (JAM-A WT Neg. ctrol. vs. miR-449a mimic) (n = 9 independent wells, 4 independent transfections).


G , H . Confocal of coronal sections from E14 brains of wild type mice stained with anti - JAM - A antibody ( green ) , phalloidin to label actin ( gray ) DAPI to label nuclei ( blue ) . Scale bars : 10 μm ( G ) , 5 μm ( H ) . I . of JAM - A expression in ventricular zone of E14 brain coronal sections from miR - 34 / 449 KO mice compared with littermate controls ( Het ) by immunofluorescence . ACTIN ( phalloidin signal ) was used to normalize ( norm . ) expresion of JAM - A . p = 0 . 04842 ( n = 5 ( KO ) or 7 ( het ) cortices per genotype group , 3 independent litters ) .

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

G, H. Confocal images of coronal sections from E14 brains of wild type mice stained with anti-JAM-A antibody (green), phalloidin to label actin (gray) and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H).I. Quantification of JAM-A protein expression in the ventricular zone of E14 brain coronal sections from miR-34/449 KO mice compared with littermate controls (Het) by immunofluorescence. ACTIN (phalloidin signal) was used to normalize (norm.) the expresion levels of JAM-A. p = 0.04842 (n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters).


A , D . Confocal time lapse of metaphase HeLa stably expressing a centriole marker ( Centrin2 - GFP ; green ) , microtubule marker ( α - tubulin - mRFP ; red ) no extra ( A ) or mouse JAM - A ( D ) , 48 h after transfection of miR - 449a mimic , JAM - A siRNA , or non - targeting negative control siRNA . Scale bar , 10 μm . B , E . of spindle rotation for as shown in A D . Each dot represents a single . Red bar indicates median . Significance was tested by Pairwise t - with Bonferroni correction . p - value = 2e - 11 ( Neg . control versus miR449 mimic ) , p - value = 0 . 01928 ( Neg . control versus JAM - A siRNA ) , p - value = 0 . 17492 ( Neg . control versus miR449 mimic , under JAM - A overexpression ) . * indicates p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; p * * * p ≤ 0 . 001 for panels . C , F . Cumulative histograms of mitotic duration from envelope breakdown until anaphase onset , measured for as shown in A D ; n ≥ 20 for conditions , 2 independent replicates .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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

A, D. Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker (Centrin2-GFP; green), the microtubule marker (α-tubulin-mRFP; red) and no extra protein (A) or mouse JAM-A (D), 48 h after transfection of miR-449a mimic, JAM-A siRNA, or non-targeting negative control siRNA. Scale bar, 10 μm.B, E. Quantification of spindle rotation for cells as shown in A and D. Each dot represents a single cell. Red bar indicates median. Significance was tested by Pairwise t-test with Bonferroni correction. p-value = 2e-11 (Neg. control versus miR449 mimic), p-value = 0.01928 (Neg. control versus JAM-A siRNA), p-value = 0.17492 (Neg. control versus miR449 mimic, under JAM-A overexpression). * indicates p ≤ 0.05; ** p ≤ 0.01; p*** p ≤ 0.001 for all panels.C, F. Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in A and D; n ≥ 20 cells for all conditions, 2 independent replicates.


B . DIS3 expression in total lysates of control ( siCTRL ) DIS3 U2OS silenced ( siDIS3 ) was analyzed by Western blot upon rescue with empty vector ( EV ) or DIS3 expressing vector ( DIS3 ) . Lamin B was used as loading control .

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B. DIS3 expression in total cell lysates of control (siCTRL) and DIS3 U2OS silenced cells (siDIS3) was analyzed by Western blot upon rescue with empty vector (EV) or DIS3 expressing vector (DIS3). Lamin B was used as the loading control.


C . Representative of comet assays performed under alkaline conditions in U2OS transfected with siCTRL or siDIS3 then infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Quantitative of comet tail lengths for each condition . Plots represent three biological replicates , with more than 150 for each point . is performed using Casplab software , error bars indicate s . e . m . ( n = 3 ) . * * * * p - value < 0 . 0001 . ns = not statistically significant , two - way ANOVA , scale bar is 200 µm .

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C. Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Quantitative analysis of comet tail lengths for each condition. Plots represent three biological replicates, with more than 150 cells for each point. The analysis is performed using Casplab software, error bars indicate s.e.m. (n=3). **** p-value < 0.0001. ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.


D . Immunostaining against RPA in U2OS transfected with siCTRL or with siDIS3 48 hours later infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . averages of foci number per were calculated from three independent experiments using Profiler software . At least 150 were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * p - value < 0 . 05 . ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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D. Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software. At least 150 cells were counted for each point, error bars indicate s.e.m. (n=3). * p-value < 0.05. ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.


A . Immunostaining with S9 . 6 ( green ) DAPI ( blue ) in U2OS , transfected for 48 hours with siCTRL or siDIS3 then mock - treated ( UNTR ) or treated with RNase H or RNase III for 1 hour before fixation . Histograms show S9 . 6 signal per , error bars indicate s . e . m . ( n = 3 , biological replicates ) . More than 150 were counted , error bars indicate s . e . m . ( n = 3 biological replicates ) . ) . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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A. Immunostaining with S9.6 (green) and DAPI (blue) in U2OS cells, transfected for 48 hours with siCTRL or siDIS3 and then mock-treated (UNTR) or treated with RNase H or RNase III for 1 hour before fixation. Histograms show S9.6 signal intensity per cell, error bars indicate s.e.m. (n=3, biological replicates). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates).). **** = p < 0.0001, *** = p < 0.001, ns = not statistically significant, two-way ANOVA, scale bar is 10 µm.


B . Immunostaining with J2 ( green ) DAPI ( blue ) in U2OS , as in panel A ) . More than 150 were counted , error bars indicate s . e . m . ( n = 3 biological replicates ) . For conditions , differences between siCTRL siDIS3 were not statistically significant , two - way ANOVA , scale bar is 10 µm .

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B. Immunostaining with J2 (green) and DAPI (blue) in U2OS cells, as in panel A). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates). For all conditions, differences between siCTRL and siDIS3 were not statistically significant, two-way ANOVA, scale bar is 10 µm.


C . Dot blot of : hybrids formation : serial dilutions ( 750 ng , 375 ng , 187 , 5 ng , 93 , 75 ng , 46 , 87 ng ) of genomic extracted from WT or DIS3 silenced U2OS pre - treated with RNase H , RNase III or mock treated , were arrayed on a probed using S9 . 6 antibody . histogram shows S9 . 6 signal in 3 biological replicates , error bars indicate s . e . m . , normalized on SYBR ® - Gold signal ( Adj . Vol . ) , using Image lab software . * * * = p < 0 . 001 , * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA .

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C. Dot blot analysis of DNA:RNA hybrids formation: serial dilutions (750 ng, 375 ng, 187,5 ng, 93,75 ng, 46,87 ng) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre-treated with RNase H, RNase III or mock treated, were arrayed on a membrane and probed using the S9.6 antibody. The histogram shows the S9.6 signal quantification in 3 biological replicates, error bars indicate s.e.m., normalized on SYBR®-Gold signal (Adj. Vol.), using Image lab software. *** = p < 0.001, ** = p < 0.01, ns= not statistically significant, two-way ANOVA.


D . DRIP - qPCR at four different R - loops sites , RPL13A , ACTB , ENSA , TRIM33 in U2OS transduced with shScr or shDIS3 treated or not with RNase H . Results are shown as mean + s . e . m of three independent experiments . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , * = p < 0 . 05 , two - way ANOVA .

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D. DRIP-qPCR analysis at four different R-loops sites, RPL13A, ACTB, ENSA, and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H. Results are shown as mean + s.e.m of three independent experiments.**** = p < 0.0001, *** = p < 0.001, * = p < 0.05, two-way ANOVA.


E . Representative pictures of comet assays performed under alkaline conditions in U2OS transfected for 48 hours with siCTRL or siDIS3 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 for each . * * * * = p < 0 . 0001 , ns = not statistically significant , two - way ANOVA , scale bar is 200 µm .

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E. Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001, ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.


A . U2OS were transfected with siCTRL or with siDIS3 48 hours later were irradiated ( IR ) or left untreated ( NO IR ) . were fixed 4 hours after IR stained for RAD51 . Histograms show foci number per , analyzed with Profiler . average number of foci per was calculated from three independent experiments . At least 200 were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * = p < 0 . 05 , two - way ANOVA , ns = not statistically significant , scale bar is 10

O O O O O O O O O O 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 O 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. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for RAD51. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). * = p < 0.05, two-way ANOVA, ns= not statistically significant, scale bar is 10 µ


D . Representative pictures of alkaline comet assays performed in U2OS silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 , at sequential time points after exposure to 3 Gy irradiation . Relative comet tail moments were plotted for siCTRL ( black circles ) siDIS3 ( red circles ) . was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with more than 150 for each siRNA . * * * * = p < 0 . 0001 , * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 20 µm .

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 O O O O O O O O O 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. Representative pictures of alkaline comet assays performed in U2OS silenced (siDIS3) or not (siCTRL) for DIS3, at sequential time points after exposure to 3 Gy irradiation. Relative comet tail moments were plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with more than 150 cells for each siRNA. **** = p < 0.0001, ** = p < 0.001, ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.


F . Representative of HPRT negative mutants in HT1080 infected with scramble shRNA ( shScr ) or shRNA against DIS3 ( shDIS3 ) . Histograms show mutation frequency deletion of DIS3 WT ( shScr ) DIS3 depleted ( shDIS3 ) after I - SceI induced DSB . Error bars represent s . e . m . ( n = 3 ) . * * * = p < 0 . 001 , ns = not statistically significant Student t - .

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F. Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA (shScr) or shRNA against DIS3 (shDIS3). Histograms show the mutation frequency and the deletion length of DIS3 WT (shScr) and DIS3 depleted cells (shDIS3) after I-SceI induced DSB. Error bars represent s.e.m. (n=3). *** = p < 0.001, ns= not statistically significant Student t-test.


A . U2OS were transfected with siCTRL or with siDIS3 48 hours later were 3 Gy irradiated ( IR ) or left untreated ( NO IR ) . were fixed 4 hours after IR stained for BRCA1 . Histograms show foci number per , analyzed with Profiler . average number of foci per was calculated from three independent experiments . At least 200 were counted for each point , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for BRCA1. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3 biological replicates). ** = p < 0.01, ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.


C . Representative of BRCA1 on U2OS transfected with siCTRL or with siDIS3 . 48 hours after transfection were irradiated 4 hours later were treated with RNase H or left untreated ( UNTR ) . Histograms show average foci number per from three independent experiments analyzed with Profiler . At least 200 were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * * = p < 0 . 01 , ns = not statistically significant , one - way ANOVA , scale bar is 10 µm .

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C. Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3. 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated (UNTR). Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). ** = p < 0.01, ns= not statistically significant, one-way ANOVA, scale bar is 10 µm.


D . Representative pictures of comet assays performed under alkaline conditions in U2OS , silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 untreated ( UNTR ) or treated with RNase H ( RNase H ) . Relative comet tail moments are plotted for siCTRL ( black circles ) siDIS3 ( red circles ) . was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 for each siRNA . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 20 µm .

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D. Representative pictures of comet assays performed under alkaline conditions in U2OS cells, silenced (siDIS3) or not (siCTRL) for DIS3 untreated (UNTR) or treated with RNase H (RNase H). Relative comet tail moments are plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each siRNA. **** = p < 0.0001, one-way ANOVA, scale bar is 20 µm.


A . Representative confocal upon irradiation with 405nm . U2OS were presensitized with BrdU for 24 hours , micro - irradiated , fixed after 10 minutes , stained with antibodies against γH2AX DIS3 scale bar is 10 µm . B . Magnification image of box in panel A . C . profiles of γH2AX ( green ) DIS3 signals through yellow in panel B . is performed with ImageJ software scale bar is 10 µm .

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A. Representative confocal images upon irradiation with the 405nm laser. U2OS cells were presensitized with BrdU for 24 hours, micro-irradiated, fixed after 10 minutes, and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm. B. Magnification image of the white box in panel A. C. Intensity profiles of γH2AX (green) and DIS3 signals through the yellow line in panel B. Quantification is performed with ImageJ software scale bar is 10 µm.


A . Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM lines MM . 1S RPMI - 8226 ( RPMI ) , in DIS3 mutated MM lines PCM6 OPM2 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 for each . * * * * = p < 0 . 0001 ns = not statistically significant , two - way ANOVA , scale bar is 20 µm .

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A. Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM.1S and RPMI-8226 (RPMI), and in DIS3 mutated MM cell lines PCM6 and OPM2, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001 ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.


B . Immunostaining with S9 . 6 ( green ) DAPI ( blue ) in MM . 1S , RPMI - 8226 , PCM6 , OPM2 , treated ( RNase H ) or not ( UNTR ) with RNase H for 1 hour . Histograms show S9 . 6 signal per . More than 150 were counted , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 10 µm .

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B. Immunostaining with S9.6 (green) and DAPI (blue) in MM.1S, RPMI-8226, PCM6, and OPM2 cells, treated (RNase H) or not (UNTR) with RNase H for 1 hour. Histograms show S9.6 signal intensity per cell. More than 150 cells were counted, error bars indicate s.e.m. (n=3 biological replicates). **** = p < 0.0001, one-way ANOVA, scale bar is 10 µm.


A . Relative mRNA expression level of interferon genes IFI6 , ISG15 , IFITM1 , IFIT3 , IFI27 , MCL1 , cGAS , STING in OPM2 infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Values are relative to housekeeping gene expression , normalized on EV , represent SE of three biological replicates . * * * = p < 0 . 001 , * * = p < 0 . 01 , multiple t - .

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A. Relative mRNA expression level of interferon genes IFI6, ISG15, IFITM1, IFIT3, IFI27, MCL1, cGAS, and STING in OPM2 cells infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Values are relative to housekeeping gene expression, normalized on EV, and represent SE of three biological replicates. *** = p < 0.001, ** = p < 0.01, multiple t-tests.


Differentially expressed genes ( DEGs ) between mutant WT samples ; p - values are from limma false - discovery rate ( FDR ) is calculated using Benjamini - Hochberg procedure ; FDR < 0 . 05 was used as cut off . Functional annotation by IPA of DEGs between DIS3 mutant WT samples on FDR cut - off . ( C ) Volcano plot of DEGs between mutant WT samples ; labeled genes are IFN - related genes with an FDR < 0 . 05 ( red horizontal ) absolute log 2 - fold change above or below 0 . 5 ( red vertical lines ) , respectively ; additional representative IFN - related genes are also labeled .

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Differentially expressed genes (DEGs) between mutant and WT samples; p-values are from limma and false-discovery rate (FDR) is calculated using the Benjamini-Hochberg procedure; FDR < 0.05 was used as cut off. Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut-off. (C) Volcano plot of DEGs between mutant and WT samples; labeled genes are the IFN-related genes with an FDR < 0.05 (red horizontal line) and absolute log 2-fold change above or below 0.5 (red vertical lines), respectively; additional representative IFN-related genes are also labeled.


( F ) Violin plot of number of non - synonymous ( NS ) mutation load comparison between DIS3 mutant versus DIS3 WT performed on entire CoMMpass dataset , including 853 MM , 88 presenting DIS3 mutations ( samples ) on CoMMpass dataset , stratified according to of IFN responsiveness ( clusters C1 , C2 , C3 ) . Wilcoxon rank - sum was used for comparison between DIS3 mutant vs WT . * * * * = p < , * * = p < 0 . 01 , * = p < 0 . 05 . Black color dots represent median in each group .

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(F) Violin plot of the number of non-synonymous (NS) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset, including 853 MM patients, 88 presenting DIS3 mutations (all samples) and on the CoMMpass dataset, stratified according to the intensity of the IFN responsiveness (clusters C1, C2, C3). Wilcoxon rank-sum test was used for comparison between DIS3 mutant vs WT. **** = p <, ** = p < 0.01, * = p < 0.05. Black color dots represent the median in each group.


( A , B ) qRT - PCR of IL6 expression upon indicated stimulations in presence or absence of NOD1 ( A ) or NOD2 ( B ) . HeLa inducible NOD1 ( A ) or NOD2 ( B ) were induced in absence or presence of doxycycline overnight afterwards stimulated with various stimuli for 4 h .

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(A, B) qRT-PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 (A) or NOD2 (B). HeLa inducible NOD1 cells (A) or NOD2 cells (B) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h.


( C ) qRT - PCR of Cxcl2 expression upon indicated stimulations in WT , Nod1 / 2 KO Rip2 KO bone marrow derived macrophages ( BMDMs ) .

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(C) qRT-PCR analysis of Cxcl2 expression upon indicated stimulations in WT, Nod1/2 KO and Rip2 KO bone marrow derived macrophages (BMDMs).


ELISA of IL6 in supernatants of HDFs ( E ) upon indicated stimulations for 20 h in absence or presence of inhibitor of sphingosine kinases .

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ELISA analysis of IL6 in supernatants of HDFs (E) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases.


( H ) qRT - PCR of Cxcl2 expression in WT , Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h .

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(H) qRT-PCR analysis of Cxcl2 expression in WT, Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h.


( D ) Western blot of MAPKs NF - κB activation upon S1P stimulation . HeLa NOD1 or NOD2 were treated with S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) for indicated times .

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(D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times.


Multiplex of CXCL2 ( I ) , CCL5 ( J ) IL16 ( K ) production in supernatants of WT , Nod1 / 2 Rip2 KO BMDMs upon S1P ( 10 µM ) or MDP ( 10 µM ) stimulations together with digitonin ( 2 . 5 µg / ml ) for 20 h .

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Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.


( A , B ) Immunoprecipitation with various lipid - coated beads using HeLa NOD1 - GFP ( A ) NOD2 - GFP ( B ) . lysates of HeLa NOD1 or HeLa NOD2 were incubated with lipid - coated beads at room for 2 h .

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(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h.


( E , F ) Single particle tracking of NOD1 - GFP ( E ) or NOD2 - GFP ( F ) with S1P - TAMRA . After doxycycline induction overnight , HeLa NOD1 or NOD2 were stimulated with S1P - TAMRA ( 20 µM ) for 1 h together with digitonin . Scale bar : 5 µm .

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(E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.


( D ) MST of direct binding of NOD1 to S1P iE - DAP . ( E ) MST of direct binding of NOD2 with S1P MDP .

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(D) MST analysis of direct binding of NOD1 to S1P and iE-DAP. (E) MST analysis of direct binding of NOD2 with S1P and MDP.


( F ) ELISA of IL8 in supernatants of HEK293T NOD1 / 2 KO transfected with indicated NOD1 or NOD2 mutants . After 24 h transfection , were stimulated with S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) together with digitonin for 20 h .

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(F) ELISA analysis of IL8 in supernatants of HEK293T NOD1/2 KO cells transfected with indicated NOD1 or NOD2 mutants. After 24 h transfection, cells were stimulated with S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) together with digitonin for 20 h.


( A , B ) MST of direct binding of NOD1 ( A ) or NOD2 ( B ) with ADP in presence of S1P ( 2 µM ) .

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(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM).


Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . were stained for γH2AX 53BP1 foci , acquired analysed using automated ‐ imaging system Hermes WiScan ImageJ . On average 104 were acquired analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ ) . Scale bars , 10μm .

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Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.


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MDM were treated with increasing concentrations of ETO CTH for 66h . were stained to distinguish between live dead using LIVE / DEAD fixable Dead stain protocol . Percentages of live / dead were determined using automated ‐ imaging system Hermes WiScan ImageJ . Addition of 20 % ethanol for 10min into treated with ETO was used as positive control . ( n = 3 , mean ± s . e . m ) .

MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).


MDM were treated with 5μM ETO for 18h infected in presence of ETO with VSV - G pseudotyped HIV - 1 GFP wild type ( wt ) HIV - 1 capsid mutants ( N74D or P90A ) , HIV - 2 , SIV sooty mangabey ( SIVsm E543 ) , replication competent Adenovirus type 5 AdV ( AdV ) , Semliki Forest ( SFV ) HSV - 1 . percentages of infected were determined using an automated ‐ imaging system Hermes WiScan ImageJ normalized to untreated control ~ 100 % . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 ; ( ns ) non ‐ significant , paired t ‐ ) . from a representative donor were used for immunoblotting .

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MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.


MDM were treated with 5μM ETO for 18h infected in presence of ETO with VSV - G pseudotyped HIV - 1 GFP . 104 were recorded analysed for GFP expression 48h post - infection using an automated ‐ imaging system Hermes WiScan ImageJ . ( n = 4 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ ) .

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MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h infected in presence of ETO with full - replication competent macrophage tropic HIV - 1 BaL . were stained for intracellular p24 of infected was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ ) .

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MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h infected in presence of ETO with HIV - 1 BaL isolated 18 h post ‐ infection for qPCR . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ ) .

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G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.  Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h infected in presence of ETO with HIV - 1 BaL isolated 18 h post ‐ infection for qPCR . 2LTR circles . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 , paired t ‐ ) .

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G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h infected in presence of ETO with HIV - 1 BaL isolated 18 h post ‐ infection for qPCR . Integrated copies of viral , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ ) .

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G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / ) . were stained for intracellular p24 of infected was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ ) .

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MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / ) isolated 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL isolated 4 , 6 , 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


MDM were transfected with control or pool of SAMHD1 ( KD ) siRNA 3 days later treated with 5μM ETO infected in presence of ETO with VSV ‐ G pseudotyped HIV ‐ 1 GFP 18h later . from a representative donor were used for immunoblotting . of infected was quantified by automated ‐ imaging system Hermes WiScan ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with VSV ‐ G HIV ‐ 1 GFP SIVmac ‐ like particles containing Vpx / Vpr ( SIV VLP ) . from a representative donor were used for immunoblotting . of infected was quantified by Hermes WiScan ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with VSV ‐ G HIV ‐ 1 GFP SIVmac ‐ like particles containing Vpx / Vpr ( SIV VLP ) or deleted for Vpx ( SIV VLP del Vpx ) or deleted for Vpr ( SIV VLP del Vpr ) . from a representative donor were used for immunoblotting . of infected was quantified by automated ‐ imaging system Hermes WiScan ImageJ 48h post - infection . ( n = 4 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIVmac ‐ like particles containing Vpx / Vpr ( SIV VLP ) . was isolated 18 h post ‐ infection for qPCR of F . Late RT products ; G . 2LTR circles ; H integrated viral . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ ) .

O O O O O 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 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 O O O O O O O O O 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-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIVmac ‐ like particles containing Vpx / Vpr ( SIV VLP ) . was isolated 18 h post ‐ infection for qPCR of F . Late RT products ; G . 2LTR circles ; H integrated viral . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ ) .

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

D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIVmac ‐ like particles containing Vpx / Vpr ( SIV VLP ) . was isolated 18 h post ‐ infection for qPCR of F . Late RT products ; G . 2LTR circles ; H integrated viral . Integrated copies of viral , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) .

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

D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


of infected was quantified by Hermes WiScan ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) .

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

The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


A - B . MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with VSV ‐ G HIV ‐ 1 GFP SIVmac ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) . MDM were differentiated cultured in Human serum instead of FCS .

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

A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in Human serum instead of FCS.


A - B . MDM were treated with 5μM ETO for 18h co ‐ infected in presence of ETO with VSV ‐ G HIV ‐ 1 GFP SIVmac ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) . MDM were differentiated cultured in FCS . A standard culture condition used in experiments . See Materials Methods .

O O O O O O O O O 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 B-CONTROLLED_VAR 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 O O O 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-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods.


C - F . MDM we treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIV VLP Q76A . was isolated 18 h post ‐ infection for qPCR of D . Late RT products ; E . 2LTR circles ; F . integrated viral . of infected was quantified by automated ‐ imaging system Hermes WiScan ImageJ 48h post - infection . from a representative donor were used for immunoblotting . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.  The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIV VLP Q76A . was isolated 18 h post ‐ infection for qPCR of D . Late RT products ; E . 2LTR circles ; F . integrated viral . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIV VLP Q76A . was isolated 18 h post ‐ infection for qPCR of D . Late RT products ; E . 2LTR circles ; F . integrated viral . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h co ‐ infected in presence of ETO with HIV - 1 BaL SIV VLP Q76A . was isolated 18 h post ‐ infection for qPCR of D . Late RT products ; E . 2LTR circles ; F . integrated viral . Integrated copies of viral , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


MDM were treated with 5μM ETO for 18h , 3μg / ml cGAMP 10ng / ml IFNβfor 18h . was isolated qPCR performed for selected genes using TaqMan assays . Expression of target genes were normalised to GAPDH . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; ( ns ) non ‐ significant , paired t ‐ ) .

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

MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO or cGAMP for 18h or 100ng / ml LPS for 2h . were stained analysed for IRF3 translocation into nucleus . Scale bars : 10μm .

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

MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.


of nuclei positive for IRF3 . ( n = 3 , mean ± s . e . m . ) .

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

Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).


MDM were treated with increasing concentrations of ETO CTH . were stained for MCM2 expression , acquired analysed using automated ‐ imaging system Hermes WiScan ImageJ . On average 104 were acquired . ( n = 3 , mean ± s . e . m . ) .

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

MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).


MDM were treated with 5μM ETO , lysed immunoblotting performed to detect cycle / cycle arrest damage associated .

MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


MDM were treated with 5μM ETO or 0 . 01μM CTH for 18h , lysed immunoblotting performed to detect cycle / cycle arrest damage associated .

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

MDM were treated with 5μM ETO or 0.01μM CTH for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


( F ) Representative of germ immunostained for SYCP3 ( green ) , DMC1 ( red ) , DDX4 ( magenta ) at each meiotic sub - stage . DAPI is shown in . sub - stages of meiosis were defined on patterns of SYCP3 , DMC1 , DAPI as described in Materials Methods . ( G ) Percentages of germ at each meiotic sub - stage as defined in Fig 1F . average percentages in outer or inner ovarian cortex on each meiotic sub - stage were calculated from at least three ovaries [ 8 wpf ( n = 4 ) , 10 / 12 / 14 / 16 / 18 wpf ( n = 3 ) ] .

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

(F) Representative images of germ cells immunostained for SYCP3 (green), DMC1 (red), and DDX4 (magenta) at each meiotic sub-stage. Nuclear DAPI staining is shown in white. The sub-stages of meiosis were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. (G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F. The average percentages in the outer or inner ovarian cortex on each meiotic sub-stage were calculated from at least three fetal ovaries [8 wpf (n=4), 10/12/14/16/18 wpf (n=3)].


( C ) IF for oogonia ( POU5F1 , TFAP2C ) , meiotic ( SYCP3 , SYCP1 ) , oogenic ( ZP3 , TP63 ) markers in transplanted cy rOvaries . Germ nuclei were marked with DDX4 ( magenta ) DAPI ( ) , respectively . upper - right magnified of each panel show expression of each marker ( green ) co - stained with DDX4 ( magenta ) . lower - right magnified of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( ) . numbers written in upper - left corner indicate after transplantation ( w - ptp ) . Scale bar = 20 μm . ( D ) Percentages of positive for markers among DDX4 + germ from IF of cy rOvaries at 3 − 15 w - ptp [ 3 w - ptp ( n = 3 ) , 6 / 9 / 12 w - ptp ( n = 4 ) , 15 w - ptp ( n = 6 ) ] . average values with SDs are shown . ( E ) H & E of transplanted cy rOvaries at 21 w - ptp . Dashed lines indicate transplanted cy rOvaries beneath kidney capsule of KSN / Slc mice . K , mouse kidney . Scale bars = 200 μm ( left ) 100 μm ( right ) .

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

(C) IF for oogonia (POU5F1, TFAP2C), meiotic (SYCP3, SYCP1), and oogenic (ZP3, TP63) markers in the transplanted cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of each marker (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate weeks after transplantation (w-ptp). Scale bar = 20 μm. (D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3−15 w-ptp [3 w-ptp (n=3), 6/9/12 w-ptp (n=4), 15 w-ptp (n=6)]. The average values with SDs are shown. (E) H&E staining of transplanted cy rOvaries at 21 w-ptp. Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN/Slc mice. K, mouse kidney. Scale bars = 200 μm (left) and 100 μm (right).


( E ) IF analyses of key markers as described in Fig 1E in cultured cy rOvaries . Germ nuclei were marked with DDX4 ( magenta ) DAPI ( ) , respectively . upper - right magnified of each panel show expression of key markers ( green ) co - stained with DDX4 ( magenta ) . lower - right magnified of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( ) . numbers written in upper - left corner indicate period of IVC ( w - ivc ) . Scale bar = 20 μm . ( F ) Percentages of positive for markers among DDX4 + germ from IF of cy rOvaries at 3 - 15 w - ivc [ 3 / 6 / 9 / 12 w - ivc ( n = 3 ) , 15 w - ivc ( n = 3 or 4 ) ] .

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 B-MEASURED_VAR 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 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

(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm. (F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)].