Fig 1: The exosome is recruited to DSBs and is necessary for HR. a HeLa cells were co-stained with antibodies against either EXOSC10 or DIS3 and ?H2AX 30 min after UV laser micro-irradiation. In some experiments, the cells were treated with Actinomycin D at low concentration (40 nM) for 1 h prior to irradiation and immunostaining to inhibit RNA polymerase I. Scale bar: 10 µm. b UV laser micro-irradiation experiments were carried out as in a, but the cells were treated with either 8 µM Actinomycin D for 1 h or 10 µM Triptolide for 30 min prior to irradiation to inhibit RNAPII. Control cells were treated with DMSO and Actinomycin D (40 nM) to facilitate the imaging of irradiated stripes. The plot shows the percentage of ?H2AX-positive stripes co-stained with antibodies against EXOSC10 or DIS3 in each condition. Statistical testing was done using a Mann–Whitney’s test and significant p-values are shown. At least 40 cells were analysed in each condition (from three independent siEXOSC10 experiments and two independent siDIS3 experiments). c Cells were transfected with either siCtrl, siEXOSC10 or siDIS3, irradiated 48 h after transfection with ionizing radiation (5 Gy), and fixed at different time-points. The percentage of cells that showed ?H2AX-positive foci were quantified. The statistical significance was tested as in b (n = 3 independent experiments). d Clonogenic assay with 0, 2, 4, and 6 Gy after EXOSC10 and DIS3 knock-down. The histogram shows cell survival 7 days after irradiation. p-values were calculated using a paired student t-test (n = 4). e, f U2OS-DR-GFP or U2OS-EJ5-GFP cells were transfected with either siCtrl, siEXOSC10 or siDIS3 and with a plasmid for I-SceI expression. GFP expression was quantified by flow cytometry and the relative repair efficiencies were calculated. The histograms show the average percentage of GFP-positive cells (% of HR repair in e and % of NHEJ repair in f) (n = 5 and n = 4 independent experiments in e and f, respectively). p-values were calculated using a two-tailed Student’s t-test. Error bars show s.e.m. Source data for Figs. 1b–f are provided as a Source Data file
Fig 2: Depletion of EXOSC10 impairs RPA and RAD51 recruitment to DSBs. a The panel shows RAD51 immunofluorescent staining of HeLa cells depleted of either EXOSC10 or DIS3 for 48 h. The cells were fixed 30 min after UV laser micro-irradiation. The bar plot in the lower part of the figure shows the percentage of γH2AX-positive stripes that were co-stained by RAD51 (n > 35 cells analysed in each condition, from two independent experiments). b RPA immunofluorescent staining was performed on cells depleted of either EXOSC10 or DIS3 and fixed 15 min after UV micro-irradiation. The bar plot shows the percentage of γH2AX-positive stripes that were co-stained by RPA (n > 60 cells analysed for each siRNA treatment, from at least two independent experiments). c CtIP immunofluorescent staining was performed on cells depleted of either EXOSC10 or DIS3 and fixed 10 min after UV micro-irradiation. The bar plot shows the percentage of γH2AX-positive stripes that were co-stained by CtIP (n > 100 cells analysed for each siRNA treatment, from two independent experiments). In all panels, the error bars represent s.e.m. Statistical testing was done using a Mann–Whitney’s test and significant p-values are shown in the figure. The scale bars represent 20 μm. Source data for Fig. 2a–c are provided as a Source Data file
Fig 3: EXOSC10 is necessary for limited DNA end resection. a HeLa cells were depleted of either EXOSC10 or DIS3, cultivated in the presence of BrdU for 24 h, UV micro-irradiated and fixed for immunofluorescence 10 min after irradiation. The bar plot shows the percentage of ?H2AX-positive stripes that were co-stained by an anti-BrdU antibody. Error bars represent s.e.m. (n > 60 cells for each siRNA treatment, from at least two independent experiments). Statistical testing was done using a Mann–Whitney’s test and significant p-values are shown in the figure. The scale bar represents 20 µm. b The image shows single-strand DNA tracks (SMART) analysed in U2OS cells 48 h after transfection with siCtrl, siEXOSC10 or siDIS3. The graph shows fibre length quantifications of one out of three biological replicates with n = 200 cells analysed. Statistical testing was done using a Mann–Whitney’s test and significant p-values are shown in the figure. c DIvA cells were transfected with either siCtrl, siEXOSC10 or siDIS3 for 48 h and treated with 300 nM 4-OHT for 4 h before extracting the genomic DNA for analysis of DNA end resection. ssDNA levels were measured by qPCR at three positions upstream of the AsiSI cleavage site (arrowhead). The non-DSB background levels were subtracted and the corrected ssDNA values are shown in the histogram. The error bars represent s.e.m. from three independent experiments. Statistical testing was done using a paired Student’s t-test (n = 3) and significant p-values are shown in the figure. The percentage of DSBs produced in each condition is shown in the figure. Source data for Fig. 3a–c are provided as a Source Data file
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