Fig. 3. The oncogenic stress triggered by high HRas^G12V activity causes DNA damage leading to cell death.

A TUNEL assays showed high levels of DNA strand breaks (DSB) in cells after six days of 4OHT induction in a dose-dependent manner. Two groups of controls were used: noninduced ER:HRas^G12V keratinocytes and ER:ø-keratinocytes treated with 50 nM of 4OHT emphasizing that the increasing levels of HRas^G12V, but not 4OHT, are responsible for DNA breaks, n = 100 cells per condition. The whole experiment (including the experimental controls and the other 4OHT concentrations used), is presented in Supplementary Fig. S4. B In ER:HRas^G12V keratinocytes, native chromatin-BrdU assay showed persistent sites of single-stranded DNA (ssDNA) after six days of 50 nM 4OHT induction. For each condition and day analyzed, at least 50 nuclei were counted and classified into three different phenotypes: normal ≤10 foci/nucleus, the initial stage of replication stress ≥10 ≤ 50 foci/nucleus, and advanced stage of replication stress ≥50 foci/nucleus. The whole kinetic experiment is displayed in Supplementary Fig. S5. C Sub-G1 populations increased with dose and time of HRas^G12V induction. The graph is representative of two independent experiments carried out in duplicate. D Annexin-V/PI-flow cytometry analyses: ER:HRas^G12V keratinocytes induced with 50 nM of 4OHT and the noninduced control condition were analyzed for 10 days. Density plots are presented as an example of the profile of ER:HRas^G12V keratinocytes in days 6 and 8. E In the same kinetic design, the treatment with 25 µM of the caspase inhibitor ZVAD did not rescue the viability of keratinocytes under high HRas^G12V activity. The graphs are representative of three independent experiments. For flow cytometry, 30 × 10³ cells were considered per condition and time point. Data presented as a mean (SD). One-way ANOVA for B and two-way ANOVA for C and D. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, Bonferroni post hoc.