Fig. 5.

Processing of ATRX-dependent HR intermediates is BLM independent. (A) HeLa and U2OS cells were transfected with siCtrl or siBLM, irradiated, and γH2AX (Left) and RAD51 (Right) foci were enumerated in EdU-negative G2 cells. Spontaneous foci (four to six γH2AX and fewer than one RAD51) were subtracted. Knockdown was confirmed by immunoblotting. (B) HeLa and U2OS cells were transfected with siCtrl or siBLM, irradiated with 2 Gy, and MUS81 foci were enumerated in pH3-positive, EdU-negative prophase cells. (C) HeLa cells were transfected with siCtrl or siBLM and incubated with BrdU for 48 h. Cells were then irradiated with 2 Gy, collected after 8 h, and processed to obtain mitotic spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Bar graph shows the IR-induced SCE numbers (total SCEs after background subtraction). Representative images of chromosome spreads from unirradiated siCtrl- and siBLM-treated cells are shown on the right; red arrows in the magnifications show individual SCE events. Individual SCE data are shown and red horizontal lines indicate the mean; 70 to 120 spreads and >4,000 chromosomes per condition were analyzed from three independent experiments. Foci and IR-induced SCE data show mean ± SEM (n = 3) and results from individual experiments, each derived from 40 (A and C) or 20 (B) cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test); NIR: nonirradiated.