Fig. 2: Distinct effects of VCP/p97 in origin firing and fork progression.
A–F DNA fiber analysis in HCT116 cells, synchronized, released for 2 h and treated for 2 h with: 10 μM VCPi, 150 μM dNTP, a combination of both or DMSO as a control (A, B); 10 μM VCPi, 20 μM CDC7i, a combination of both or DMSO (C, D); 10 μM VCPi, 0.5 μM POLAi, a combination of both or DMSO (E, F). FR (A, C, E) and 1st label origins (B, D, F) were determined. G, H DNA fiber analysis in cells synchronized, released for 2 h and treated for 2 h with: 10 μM VCPi, 0.5 μM POLAi, a combination of both or DMSO. CldU/IdU ratio (G) and long/short ratio (H) were determined. Individual data for the long/short ratio are shown in Supplementary Fig. 2B–E. I, J DNA fiber analysis in cells synchronized, released for 2 h and treated for 2 h with 0.5 μM POLAi, 20 μM CDC7i, a combination of both or DMSO. FR (I) and 1st label origins (J) were determined. For all DNA fiber analysis three independent experiments were performed; dot plots show pooled data and bars represent the median (FR, CldU/IdU and long/short fork) and bar graphs (1st label origins) plot the mean ± SD. The median of individual experiments is noted with different symbols both in dot plots and bar graphs. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant, in Kruskall–Wallis with Dunn’s post-test (A, C, E, G, H, I) or one-way ANOVA followed by Tukey’s test (B, D, F, J). K Western blot analysis of whole cell extracts from HCT116 cells, synchronized, released for 2 h and treated for 2 h with increasing concentrations of POLAi (0.1, 0.2, 0.5, 1 μM) alone or in combination with 10 μM VCPi. The levels of total and phosphorylated CHK1 (S345), total and phosphorylated RPA2 (S4/S8), phosphorylated H2AX (γH2AX) and total histone H2A were measured with specific antibodies. The experiment was repeated three times and one representative result is shown. Source data are provided as a Source Data file.
