EMBO/SourceData huggingface dataset:
figf1(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 and anti-Prc1immunoblots of lysates of the same cells.(c) DNA histograms of the same cells at the indicated time points.(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 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.
figf2(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 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) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days.(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 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 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 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.
figf3(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 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) 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) 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 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 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 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.
figf4(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.
figf5(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 the same cells with the indicated antibodies.
Figure 1A. 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 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 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 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.
Figure 2A. 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 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 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), 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 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.
Figure 3A. 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 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 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 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 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), 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).
Figure 4A. 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 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 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 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 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).
Figure 1B. 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 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 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.
Figure 2A. 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) 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 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 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 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.
Figure 3A. 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 µD. 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 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.
Figure 4A. 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 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 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.
Figure 5A. 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.
Figure 6A. 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) 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.
Figure 7A. 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 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 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.
Figure 1(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.
Figure 2(C) qRT-PCR analysis of Cxcl2 expression upon indicated stimulations in WT, Nod1/2 KO and Rip2 KO bone marrow derived macrophages (BMDMs).
Figure 3ELISA 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 analysis of Cxcl2 expression in WT, Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h.
Figure 4(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 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.