Figure 3

CHD4 depletion does not affect overall ATM DNA damage signalling following IR, but does enhance the magnitude and duration of the DNA damage response. (A) CHD4S1349 and γH2AX co-staining in unirradiated or 1 hr following 2Gy IR, in mock (siControl) and CHD4 depleted (siCHD4) HeLa cells. Dapi stained nuclei are shown in the merged image. Detergent pre-extraction was performed before immunostaining. The enlarged image shows CHD4S1349 and γH2AX co-staining. (B) HeLa cells were treated with mock (siControl) or CHD4 depleted (siCHD4), after 48 h cell lysates were prepared and subjected to western blot analysis for CHD4 and DNAPK as indicated. (C) HeLa cells were treated with mock (siControl) or CHD4 depleted (siCHD4). Cells were then treated with 10Gy IR or mock irradiated and cell lysates were prepared after 1 h. The lysates were subjected to western blot analysis for ATM S1981, P53 S15, P53, Chk1 S315 and Chk1 as indicated. (D) In-nuclear-western analysis of chromatin bound 53BP1 in mock (siControl) and CHD4 depleted (siCHD4) treated U2OS cells. Cells were treated with 4Gy IR or unirradiated (0 hr), at the indicated time points detergent pre-extraction was used to remove non-chromatin bound proteins prior to fixation and immunofluorescence analysis. Mean fluorescence signal was then calculated from at least 300 cells. (E, F) In-nuclearwestern analysis of chromatin bound γH2AX (E) and 53BP1 (F) in mock (siControl) and CHD4 depleted (siCHD4) U2OS cells treated as for (D). Graphs represent the mean of three experiments + and - the standard deviation. (G) U2OS cells were treated with mock (siControl) or CHD4 depleted (siCHD4). Cells were treated with 4Gy IR or unirradiated (0 hr), cell lysates were prepared at the times indicated and subjected to western blot analysis for ATM S1981, ATM, Smc1 S957, Kap1 S824 or CHD4 as indicated.