Fig. 6: VCP/p97 limits priming by POLA/PRIM.

Comet assay on nascent DNA labelled with EdU. Cells were incubated with 30 μM EdU for 60 min, and EdU was conjugated to a fluorescent probe after alkaline comet assay. A, B HCT116 cells were synchronized, released for 2.5 h and treated for 2 h with 0.5 μM adarotene (POLAi) or DMSO as a control. Representative images are shown in (A) and quantification in (B). C, D HCT116 cells were transfected with a non-specific siRNA (siC) or a combination of individual siRNA directed against POLA1, POLA2, PRIM1 and PRIM2. 24 h after transfection cells were synchronized, released for 3 h and incubated with EdU. Representative images are shown in (C) and quantification in (D). E, F HCT116 cells were synchronized, released for 2.5 h and treated for 2 h with 5 μM NMS873 (VCPi) or DMSO as a control. Representative images are shown in (E) and quantification in (F). G, H HCT116 cells were synchronized, released for 2.5 h and treated for 2 h with 0.5 μM adarotene (POLAi) alone or in combination with 5 μM NMS873 (VCPi). Representative images are shown in (G) and quantification in (H). I, J HCT116 cells were transfected with a combination of individual siRNA directed against POLA1, POLA2, PRIM1 and PRIM2. 24 h after transfection cells were synchronized, released for 2.5 h HCT116 cells and treated for 2 h with 5 μM NMS873 (VCPi) or DMSO as a control. Representative images are shown in (I) and quantification in (J). In all cases the dot plots show pooled data of at least 4 experiments and bars represent the median of pooled data. The individual medians of independent experiments are noted with different symbols. **p < 0.01; ****p < 0.0001; ns, non-significant, in Mann–Whitney test (B, D, F, H, J). Source data are provided as a Source Data file.