Impaired nucleotide metabolism activates p21 in a p53/p73-dependent manner. (A and B) p21 level change. HeLa shPGAM1#1 or NCI-H1299 shPGAM1#1 cells transfected with indicated siRNAs for 48 h were harvested for immunoblotting analysis. Scr, scramble cells. (C) Subcellular localization of p73 in PGAM1 knockdown HeLa cells. Nuc, nuclear fraction; Cyto, cytoplasmic fraction. (D and E) p73 enrichment in p21 promoter detected by ChIP assay. HeLa shPGAM1#1 or NCI-H1299 shPGAM1#1 cells were subjected to ChIP assay using anti-p73 antibody followed by qPCR analysis using primers targeting indicated p21 promoter region. dNTP at 100 µM was added 24 h before harvest. (F–H) p21 level change. A549 or CAL51 cells transfected with indicated siRNAs for 48 h were harvested for immunoblotting analysis. (I) Subcellular localization of p53 in PGAM1 knockdown A549 cells. (J) p53 enrichment in p21 promoter detected by ChIP assay. A549 cells transfected with indicated siRNAs for 48 h were subjected to ChIP assay using anti-p53 antibody followed by qPCR analysis as described in D. siNC, negative control siRNA. Error bars represent mean ± SD of triplicates. **, P < 0.01; ***, P < 0.001; n.s., not significant.