PGAM1 is required for HR repair of DSBs. (A and B) Kinetics of γH2AX levels and foci formation. HeLa shPGAM1#1, shPGAM1#3, or scramble (Scr) cells were treated with CPT (1 µM) for 2 h followed by drug-free culture for up to 8 h. γH2AX levels were detected by immunoblotting (A), and γH2AX foci were detected by immunofluorescence assay (B). Foci were quantified by counting at least 100 cells per sample. Bar, 10 µm. (C) Comet assay. Cells were treated as described in A before harvest for comet assay. Tail moments were quantified by measuring 50 cells per sample using CASP software. Bar, 100 µm. (D) Cell cycle analysis. HeLa shPGAM1#1, shPGAM1#2, or Scr cells were treated with CPT (10 nM) for 24 h, and the cell cycle profile was analyzed by flow cytometry. (E and F) HR and NHEJ repair assay. DR-U2OS (E) or NHEJ-HeLa (F) cells were transfected with indicated siRNAs for 24 h, followed by I-SceI transfection. KU55933 (10 µM) or NU7441 (10 µM) was added at the time of I-SceI transfection. GFP-positive cells were analyzed by flow cytometry 48 h later. Knockdown efficiency was measured by immunoblotting. siNC, negative control siRNA. Error bars represent mean ± SD of triplicates. *, P < 0.05; ***, P < 0.001.