Fig. 4: RHOJ promotes DNA repair and the activation of new origins of DNA replication after chemotherapy in EMT tumour cells.

a, Western blot analysis of phosphorylation of ATM/ATR substrates in EPCAM⁺, EPCAM⁻ and Rhoj-KO EPCAM⁻ cells untreated and treated with cisplatin/5FU for 12 h and 24 h. n = 3. b,c, Western blot analysis (b; n = 3) and FACS analysis quantification (c; n = 5) of the percentage of cells expressing γ-H2AX in EPCAM⁺, EPCAM⁻ and EPCAM⁻ Rhoj-KO cells, 12 h and 24 h after cisplatin/5FU administration. β-Actin was the loading control. n values represent the number of independent primary cultured cell lines. d,e, Representative cell cycle FACS profile (d) and quantification (e) of the percentage of cells in G0/G1, S and G2/M phase 12 h and 24 h after cisplatin/5FU treatment of EPCAM⁺, EPCAM⁻ and EPCAM⁻ Rhoj-KO BrdU-7AAD-labelled cells. n values indicate the number of independent experiments. f, Representative stretched DNA fibres labelled with CldU and IdU from EPCAM⁺, EPCAM⁻ and EPCAM⁻ Rhoj-KO cells untreated and treated with cisplatin/5FU for 12 h. n = 6. Scale bar, 10 μm. g, Fork rate (FR) values in EPCAM⁺, EPCAM⁻ and EPCAM⁻ Rhoj-KO cells untreated and treated with cisplatin/5FU for 12 h. Data are median. Statistical analysis was performed using ANOVA, with condition, experience and their interaction; the P value was adjusted using the two-way post hoc Sidak test. n values represent the number of forks pooled from six independent experiments. h, The percentage of origin of firing in EPCAM⁺, EPCAM⁻ and EPCAM⁻ Rhoj-KO cells untreated and treated with cisplatin/5FU for 12 h. From left to right, total number of DNA fibres scored: n = 3,020, 3,123, 3,065, 3,091, 3,078 and 3,069, in six independent experiments. For c, e and h, statistical analysis was performed using Kruskal–Wallis tests followed by two-sided Mann–Whitney U-tests; P values were adjusted for multiple comparisons using Bonferroni correction. For the box plots, the centre line shows the median, the box limits represent the 25th and 75th percentiles, and the whiskers show the minimum and maximum values.