Fig. 8: Catalytic activity of primase PRIM1, not PRIMPOL, is required for efficient replication restart after stress.

a Representative immunoblot in indicated U2OS cells treated with HU (0 = untreated, 24 = 24 hr treatment, R = 24 hr treatment followed by 8 hr release). α-tubulin represents loading control. b Top: Labeling schematic. PBS washes are indicated by a black vertical line in all schematics. Bottom: Ratio of CldU:IdU tract lengths in indicated U2OS cells. c Representative immunoblot in U2OS cells transfected with siRNA and treated with HU as indicated (0 = untreated, 24 = 24 hr treatment, R = 24 hr treatment followed by 8 hr release). α-tubulin represents loading control. d–f Top: Schematic of labeling scheme. PBS washes are indicated by a black vertical line in all schematics. In (f), V-TP, a potent PRIM1 inhibitor was added as indicated. Bottom: Quantification of representative experiment of CldU:IdU tract length ratio in U2OS cells after indicated perturbations, including transfuction or transfection with indicated RNAi reagents. Experiments conducted at least three times in biological replicates with consistent results for (a)–(f). Mean is shown with red line for (b), (d), (e), (f). Experiments were blinded prior to analysis for (b), (d), (e), (f). Average CldU:IdU ratios are listed above each sample for (b), (d), (e), (f). Outliers removed with ROUT (1%) for (b), (d), (e), (f). Significance evaluated by Kruskal-Wallis ANOVA with a Dunn’s post-test. RH2A = RNASEH2A, Ctrl = Control. Source data are provided as a Source Data file.