Fig. 1: VCP/p97 is necessary for DNA replication.

A, B Flow cytometry analysis of DNA content (DAPI) and DNA replication (EdU) in HCT116 cells, synchronized, released for 3 h and treated with 2 and 5 μM NMS873 (VCPi) for 3 h (A) or with 5 μM NMS873 (VCPi) for 3/6 h (B). One out of two representative experiments is shown. C–H DNA fiber analysis in HCT116 cells, synchronized, released for 2 h, treated for 2 h with 10 μM NMS873 (VCPi) or DMSO (control). C To measure fork rate (FR) and origin firing, cells were sequentially incubated with CldU and IdU for 20 min. FR was measured by the length of the IdU track. Origin firing (1^st label origins) was measured as the percentage of CldU tracks flanked by IdU tracks. D, E show FR and 1^st label origins upon treatment with 5 μM NMS873 (VCPi) for 2 h or DMSO (control, C). The pool (fork rate, bars represent the median of the pooled, ***p = 0.0006, Mann–Whitney test) or the average (1^st label origins, %, mean ± SD, **p = 0.0022, t test) of three experiments is shown. Symbols note the median of individual experiments. F For fork (CldU/IdU) and origin asymmetry (long/short), cells were sequentially incubated with CldU for 10 min and IdU for 30 min. The ratio of the track lengths CldU/IdU (CldU/IdU ratio) was used to follow fork stalling and the ratio of long and short IdU tracks arising from the same origin (long/short ratio) was used to follow origin asymmetry. G and H show the CldU/IdU ratio and long/short ratio upon treatment with 5 μM NMS873 (VCPi) for 2 h or DMSO (control, C). The pool of three independent experiments is shown. Bars represent the median of the pooled data. Symbols note the median of individual experiments. ****p < 0.0001 in (G, H), Mann–Whitney test. I Western blot analysis of whole cell extracts obtained from HCT116 cells, synchronized, released for 2 h and treated with 2 mM hydroxyurea (HU), 10 μM NMS873 (VCPi), a combination of both (HU-VCPi) or DMSO (lane 1, C). The indicated proteins were measured with specific antibodies. One of two repetitions is shown. Source data are provided as a Source Data file.