Decreased CtIP stability results from deficient dNTP synthesis. (A and B) dNTP level change. Intracellular individual dNTP levels were measured using LC-MS/MS analysis. (A) HeLa shPGAM1#1 cells reconstituted with empty vector (EV), WT, or mutant PGAM1; Scr, scramble cells. (B) HeLa cells treated with PGMI-004A (20 µM) for 24 h. (C) CtIP protein level change. HeLa shPGAM1#1 cells were treated with 100 µM dNTPs for 24 h before being subjected to immunoblotting. (D) HR repair assay. DR-U2OS cells were transfected with indicated siRNAs for 24 h followed by I-SceI transfection. dNTP (100 µM) or KU55933 (10 µM) was added at the time of I-SceI transfection. GFP-positive cells were analyzed by FACS analysis 48 h later. (E) CtIP protein level change. Cells transfected with indicated siRNAs for 72 h were harvested for immunoblotting analysis. (F) CtIP ubiquitylation assay. HeLa shPGAM1#1 or scramble (Scr) cells were transfected with FLAG-CtIP for 48 h, and MG132 (10 µM) was added 6 h before harvest. Cell lysates were subjected to immunoprecipitation using FLAG M2 beads followed by blotting with anti-ubiquitin antibody. (G and H) p21 level change. p21 mRNA or protein levels were examined using real-time PCR or immunoblotting analysis. (G) HeLa cells transfected with indicated siRNAs for 48 h. (H) HeLa shPGAM1#1 cells treated with dNTP (100 µM) for 24 h. (I) CtIP protein level change. Cells were transfected with indicated siRNAs for 48 h before being subjected to immunoblotting analysis. (J) HR repair assay. DR-U2OS cells were transfected with indicated siRNAs for 24 h. HR repair was assessed as in D. siNC, negative control siRNA. Error bars represent mean ± SD of triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.