Fig. 4: Inhibition of calcium channels increases sensitivity to MAPK inhibitors in partially reprogrammed cells.

a Cell viability was performed using alamar blue assay. Partially reprogrammed cells C790 were treated with mibefradil (10–1.25 μM) for 24 h. After 3 h incubation with alamar blue, fluorescence (at 590 nm) was obtained. Cell viability was evaluated at days 6, 12 and 20 of reprogramming. b Representative scatter plots of PI (y-axis) vs. annexin V (x-axis) for day 20 of partial reprogramming. Early apoptotic cells are shown in the lower right quadrant and late apoptotic cells are shown in the upper right quadrant. Mibe: mibefradil (7 μM). Lome: lomerizine (7 μM). Mibe (Lome) »Tra: sequential treatment with mibefradil (lomerizine) 24 h, followed by trametinib (15 μM) for another 24 h. c Apoptosis analysis using annexinV/PI staining. Cells were treated with mibefradil (7 μM) and lomerizine (7 μM) for 24 h. Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 3). d. Apoptosis analysis using sequential treatment with calcium channel blockers and MAPKi. Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 3). e Clonogenic assay of partially reprogrammed cells C790 treated with DMSO (0.01%), mibefradil (7 μM), lomerizine (7 μM), Mibe (Lome) »Tra: sequential treatment with mibefradil (lomerizine), and trametinib (15 μM) for 24 h. Representative images of wells stained with crystal violet are shown. f Percentage of colony area for all treatments is shown as mean ± SEM (n = 5) for partially reprogrammed cells C790. All data are represented as mean ± SEM of three or more independent experiments. Statistical analyses were performed with One-Way ANOVA and post-hoc test; *p < 0.05, **p < 0.01 and ***p < 0.001.