Enzymatic inhibition of PGAM1 impairs CtIP stability and DSB end resection. (A) ssDNA detection. HeLa shPGAM1#1 or scramble (Scr) cells were incubated with 20 µM BrdU for 36 h followed by CPT (1 µM) treatment for 2 h. ssDNA was detected using anti-BrdU antibody without denaturation. BrdU staining under denaturing conditions (2 M HCl) shows total BrdU incorporation. Knockdown efficiency was detected by immunoblotting. (B–D) RPA foci formation. Cells were exposed to CPT (1 µM) for 2 h, and RPA foci were detected by immunofluorescence assay. Foci were quantified by counting at least 100 cells per sample. HeLa shPGAM1#1 cells (B); HeLa shPGAM1#1 cells reconstituted with empty vector (EV), WT, or mutant PGAM1 (C); HeLa shPGAM1#1 pretreated with Me-2-PG (5 µM, 24 h; D). (E) CtIP foci formation. HeLa shPGAM1#1 cells were treated with 1 µM CPT for 2 h. CtIP foci were detected by immunofluorescence assay and quantified by counting at least 100 cells per sample. (F–I) CtIP protein level change. CtIP protein level was analyzed by immunoblotting. CtIP level was semiquantified by densitometry and normalized to untreated cells. HeLa shPGAM1#1 cells (F); HeLa shPGAM1#1 cells reconstituted with EV, WT, or mutant PGAM1 (G); HeLa cells transfected with indicated siRNAs for 48 h (H); HeLa shPGAM1#1 cells treated with CHX at 20 µM for indicated times (I). siNC, negative control siRNA. Bar, 10 µm. Error bars represent mean ± SD of triplicates. **, P < 0.01; ***, P < 0.001; n.s., not significant.