Fig. 7

ID3-depleted cells are sensitive to IR and are defective in DSB repair. a HeLa cells with a stable ID3 knockdown were exposed to the indicated doses of IR and assessed for colony forming ability (n = 3). The cell viability of untreated cells is defined as 100%. b, c γ-H2AX foci (b) and comet moments (c) at indicated time points after exposure to IR in both control and ID3-depleted HeLa cells. Representative images (upper panel) and quantification (lower panel) of unrepaired DSBs are shown (n = 3). d HeLa cells were transfected with indicated siRNA combinations, and the expression levels of ID3 and MDC1 were assessed by western blotting using antibodies indicated. e HeLa cells were transfected with indicated siRNA combinations. 1 h after exposure to IR, the cells were assessed using the comet assay. Representative images (upper panel) and quantification (lower panel) of comet tail moments are shown (n = 3). f A western blot of GFP-tagged wild-type (WT) ID3 or GFP alone (mock construct) expressed in ID3 knockdown HeLa cells is shown. g Comet moments of complemented HeLa cells are shown as in (e). ID3-depleted cells were complemented with a GFP-WT ID3 construct or with GFP alone. h A western blot of the indicated GFP-ID3 constructs reconstituted into ID3 knockdown HeLa cells is shown. i Comet moments of complemented HeLa cells are shown as in (e). ID3-depleted cells were reconstituted with either HLH ID3 or an HLH deletion mutant (ΔHLH) of ID3 (n = 3). j A western blot demonstrating comparable expression of the indicated GFP-tagged point mutants in the ID3 HLH domain expressed in ID3 knockdown HeLa cells is shown. k Similar to i but with the indicated point mutants in the ID3 HLH domain expressed in ID3 knockdown HeLa cells (n = 3). Scale bars: 40 μm (b) and 200 μm (c, e, g, i, k). Uncropped blots of this Figure are shown in Supplementary Fig. 14. Data are presented as means ± s.d. P values are based on Student’s two-tailed t-test: **P < 0.01. ns, not significant