Fig. 4: Depletion of C16orf72 leads to replication stress.
a Immuno-fluorescence microscopy quantification of nuclear γH2AX intensity in wild-type U2OS and C16orf72 knock-out cells (c16orf72Δ.2) treated with 200 µM hydroxyurea (HU) for 24 h with or without 6 h recovery thereafter. Mean % γH2AX positive cells ± SEM of 3 biological independent experiments with at least 1500 cells analysed per condition. b–d DNA fibre spreading analysis of wild-type U2OS and C16orf72∆ cells. U2OS, C16orf72∆ (c16orf72Δ.2 and c16orf72Δ.3) and C16orf72∆ cells complemented with Flag-C16orf72 (+ C16orf72) were left untreated, or exposed to 2 mM hydroxyurea (HU) for 2 h according to the schematic (upper panel). CldU/IdU labelling patterns and tract length were used to determine replication fork speed (b), percentage of DNA fibres with stalled forks (c) or new origins (d). Data presented are derived from 3 biological independent experiments. Mean values are represented +/− SEM. e Quantitative image-based cytometry (QIBC) of 53BP1 level in wild-type U2OS and C16orf72 knock-out cells (c16orf72Δ.2 and c16orf72Δ.3) left untreated or exposed to 0.2 µM aphidicolin for 24 h. Below: representative images of 53BP1 induction in the cells. Scale bars represent 20 μm. Above: mean nuclear 53BP1 bodies per G1 nucleus (Cyclin A negative) ± SEM of 3 biological independent experiments with at least 400 G1 cells (cyclin A-negative cells) analysed per condition. For all plots, mean ± SEM of three independent biological repeats shown. Statistical analysis for (b) was Mann-Whitney non-parametric. In all other instances statistical significance was determined using a Ordinary one-way ANOVA with a Bonferroni post-hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.
