Fig. 2: Oncogenic stress induced by high HRas^G12V activity in E6E7 keratocytes causes cell cycle arrest at G1/G0 and G2/M phases.

A Growth curve of ER:HRas^G12V keratinocytes in the supplemented medium induced or not with 50 nM of 4OHT. The graph is representative of three independent experiments carried out in triplicate. B Cell cycle profile analyses of growing ER:HRas^G12V keratinocytes, using 1-h pulse of EdU incorporation and propidium iodide (PI) staining, showed a progressive decrease in DNA synthesis (red arrows) in cells induced with 50 nM 4OHT. C DNA content profiles of growing ER:HRas^G12V keratinocytes stained with PI, with or without 4OHT induction. D Steady-state growing ER:HRas^G12V keratinocytes were induced or not with 50 nM 4OHT at zero time: kinetics of the relative size of cell cycle-phase subpopulations; evidence of cell cycle arrest at G1/G0 and G2/M phases only in 4OHT-induced cells. The graph is representative of four independent experiments, two PI single-labeled, and two PI plus EdU double-labeled. All conditions and time points were carried out in duplicate. E Immunoblots showed intracellular levels of critical components of cell cycle control pathways in growing ER:HRas^G12V keratinocytes induced or not with 4OHT. For flow cytometry, 30 × 10³ cells were considered per condition and time point. Data presented as a mean (SD). Two-way ANOVA *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, Bonferroni post hoc.