Fig 2: Cells lacking INO80 have a modest resection defect and a more significant defect in RAD51 foci accumulation, and these defects are rescued by depletion of H2A.Z IR induced RPA focus formation in A549 cells treated with siControl, siINO80, siH2A.Z or siINO80/siH2A.Z. Left hand panel: representative images. Right hand panel: quantification of foci. IR induced RAD51 focus formation in A549 cells treated with siControl, siINO80, siH2A.Z or siINO80/siH2A.Z. Left hand panel: representative images. Right hand panel: quantification of foci. Western blot analysis showing efficiency of siRNA depletion. KAP1 is used as a loading control. Quanitification of RPA foci following 1 h of treatment with camptothecin (CPT). Data information: Scale bars represent 5 μm. Data represent the mean of 4 (A, B) or 3 (D) independent assays ± SD. NS, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by Student’s t-test.

Fig 3: ARP8 phosphorylation deficiency increased its interaction with INO80.(A) Immunoprecipitation analysis of the interaction between INO80 and ARP8 in ATM inhibitor treated U2OS cells. The cells were treated with 10 μM KU55933 (ATMi) or equal amounts of DMSO (mock) for 2 hr, and then treated with DMSO (ctrl) or etoposide for 15 min, washed, and then cultured in fresh medium with or without 5 μM KU55933 for 2 hr. Immunoprecipitation analysis was performed with either anti-ARP8 antibodies or normal IgG. The relative immunoprecipitated amounts of INO80 are shown. Quantitative analysis was performed using the Image J software. (B) Examination of the interaction between INO80 and ARP8 in U2OS cells expressing HA-tagged wild-type or S412A ARP8. The endogenous ARP8-depleted cells were treated with etoposide for 15 min. After the cells were washed, they were placed in fresh medium and harvested at the indicated time points. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-INO80 or an anti-HA or an anti-ATM/ATR substrate antibody. The amounts of INO80 and HA-ARP8 were quantified, using the Image J software. The results of quantitative analysis are shown as the relative values as compared to the DMSO control from three independent experiments. Source data are presented in Figure 3—source data 1.10.7554/eLife.32222.014Figure 3—source data 1.Source raw data for Figure 3B.10.7554/eLife.32222.015Figure 3—source data 2.Source raw data for Figure 3—figure supplement 1B.10.7554/eLife.32222.016Figure 3—source data 3.Source raw data for Figure 3—figure supplement 2B.

Fig 4: INO80 and ANP32E work together to promote RAD51 foci formation following irradiation, which is rescued by the depletion of H2A.Z IR induced RPA focus formation in A549 cells treated with siControl, siANP32E, siANP32E/siINO80 or siANP32E/siH2A.Z. Left hand panel: representative images. Right hand panel: mean number of foci ± SD. IR induced RAD51 focus formation in A549 cells treated with siControl, siANP32E, siANP32E/siINO80 or siANP32E/siH2A.Z. Left hand panel: representative images. Right hand panel: mean number of foci ± SD. Western blot analysis showing efficiency of siRNA depletion. Ku80 is used as a loading control. Data information: Scale bars represent 5 μm. All data (A, B) are from a minimum of 3 independent assays. NS, not significant; *P ≤ 0.05; ***P ≤ 0.001 by Student’s t-test.

Fig 5: H2A.Z dynamics at damaged chromatin H2A.Z is rapidly incorporated and removed from chromatin in the vicinity of damaged DNA. U2OS cells transfected with GFP-H2A.Z were laser micro-irradiated and monitored by live cell imaging. Representative images taken at indicated time points are shown. The RuvBL2 subunit of INO80 accumulates at sites of damaged DNA. U2OS cells transfected with EGFP-RuvBL2 were laser micro-irradiated and monitored by live cell imaging. Representative images taken at indicated time points are shown. Quantification of mean fluorescence intensity at sites of micro-irradiation ± SE (n = 3). Removal of H2A.Z from chromatin in the vicinity of damaged DNA is at least partly dependent on INO80. USOS cells treated with siINO80 were transfected with GFP-H2A.Z, laser micro-irradiated and monitored by live cell imaging. Representative images taken at indicated time points are shown. Quantification of relative mean fluorescence intensity ± SD at sites of micro-irradiation in control (U2OS; n = 3) and siINO80 cells (n = 3) in a minimum of 10 cells per experiment. Data information: Scale bars represent 10 μm.