PGAM1 enzymatic activity is required for HR repair. (A and B) Kinetics of γH2AX levels and foci formation. PGAM1 stable knockdown cells (HeLa shPGAM1#1) reconstituted with empty vector (EV), WT, or mutant PGAM1 were treated with CPT (1 µM) for 2 h, followed by drug-free culture for up to 8 h, and subjected to immunoblotting (A) or immunofluorescence (B) assay. Foci were quantified by counting at least 100 cells per sample. Bar, 10 µm. (C) HR repair assay. DR-U2OS cells were pretreated with methyl-2-PG (Me-2-PG, 5 µM) or PGMI-004A (20 µM) for 24 h followed by I-SceI transfection. KU55933 (10 µM) was added at the time of I-SceI transfection. GFP-positive cells were analyzed by flow cytometry 48 h later. (D and E) Cell apoptosis assay. PGAM1-reconstituted cells as described in A or HeLa shPGAM1#1 cells were pretreated with Me-2-PG (5 µM) for 24 h. Apoptosis was then induced by CPT treatment for 48 h followed by Annexin V–PI dual staining. (F) Cell viability assay. HeLa cells were treated with CPT or CDDP alone or in combination with PGMI-004A (20 µM) for 72 h. Cell viability was measured by Sulforhodamine B assay. (G and H) HR repair assay. DR-U2OS cells were transfected with indicated siRNAs for 24 h followed by I-SceI transfection as in C. Knockdown efficiency was measured by immunoblotting. Error bars represent mean ± SD of triplicates. ***, P < 0.001; n.s., not significant.