Fig. 3: Inhibition of integrative stress response and ROS production does not affect apoptosis induction by P2Et on B16-F10 cells.

B16-F10 cells were 2 h pre-treated with salubrinal or ISRIB and then treated with P2Et IC50 (74.7 µg/ml) or Vehicle for additional 24 h. a A representative image of eIF2a total or p-eIF2α analysis by western blot of B16-F10 cells pretreated with several concentrations of salubrinal (10, 25, 50, and 75 µM). β-actin was used as a loading control. b A representative contour plot of B16-F10 cells pretreated with 75 µM Salubrinal, treated with P2Et or Vehicle and labeled with Annexin V-FITC and PI is shown. c Percentages of Annexin V positive cells were expressed as mean ± SEM of three independent experiments. d A representative contour plot of B16-F10 cells pretreated with several concentrations of ISRIB (1, 2, and 5 µM) and treated with P2Et or vehicle for additional 24 h. e Percentages of Annexin V positive cells were expressed as mean ± SEM of three independent experiments. f B16-F10 cells were treated with P2Et IC50 or Vehicle for 6, 12, and 24 h, following cells were harvested and labeled with 100 mM CellROX green. A representative histogram is shown. g Percentage folding change of CellROX MFI from treated cells relative to the vehicle from three independent experiments is shown. h B16-F10 cells were pre-treated 2 h with antioxidants (2 mM mitoTEMPO, 2 mM sulforaphane, and 2.5 mM N-acetyl-cysteine-NAC), and then treated with P2Et IC50 (74.7 µg/ml) or Vehicle for additional 24 h. A representative contour plot of B16-F10 cells stained with Annexin V-FITC and PI is shown. i Percentages of Annexin V positive cells were expressed as mean ± SEM of three independent experiments. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001