Dose response and proliferative effect of verapamil in MIN6 cells. To determine the proliferative effect of verapamil, (A) MIN6 cells were treated with various concentrations of verapamil (μM) for 24 and 48 h in high glucose supplemented DMEM media as described in Section 2. Cell viability percentages were calculated relative to the untreated controls. (B) MIN6 cells were cultured in the high glucose supplemented DMEM media in the presence of verapamil (1–50 μM for 24 h), and relative cell viability percentages were calculated for untreated cells. (C,D) MIN6 cells were cultured on coverslips in high-glucose DMEM media supplemented with 2% free fatty acid BSA. The cells were treated with 50 μM of verapamil for 24 h. Then Ki67 immunostaining was conducted as described in Section 2. Cell compartments were stained as nuclei (blue—DAPI), actin filaments (red—phalloidin), and Ki67 positive proliferating cells (green). Nuclear co-localization is shown by yellow arrows (bar: 50 μm, n = 2). The corrected total cell fluorescence (CTCF) was calculated from 10 fields per sample. (E,F) Western blotting experiment using proteins extracted from MIN6 cells cultured in high-glucose DMEM media with 2% free fatty acid BSA, treated with 50 μM of verapamil for 24 h to detect the level of histone H3 protein expression as another proliferation marker compared to untreated cells. The level of histone H3 protein was normalized to β-Aactin (n = 2). These data showed no significant changes in relative cell viability, Ki67, and histone H3 protein expressions. (G,H) MIN6 cells cultured in high-glucose DMEM media with 2% free fatty acid BSA treated with 50 μM of verapamil for 24 h showed significantly higher CCK and PCNA protein expressions relative to β-Actin detected by Western blotting assay (n = 3). (I) MIN6 cells were cultured in DMEM media supplemented with FFA-free 2% BSA for 24 h. Then, the cells were treated with verapamil (50 μM) for 1, 2, 3, or 4 h, or left untreated as control. Harvested single cells were fixed with ice-cold 70% ethanol, and after centrifugation, the cells were treated at room temperature (RT) with RNase (100 µg/mL) for 30 min, followed by incubation with PI (40 µg/mL) for another 30 min. DNA content and cell cycle were analyzed using BD FACSCant Flow Cytometer, as described in detail in Section 2. Flow cytometry analysis tracked verapamil’s time-dependent effect on cell cycle phases: dead cells (black), G0/G1 (blue), S-phase (gray), and G2/M (red) cell populations (n = 4 independent experiments). (J) MIN6 cells were cultured at a concentration of 5 × 104 cells/well in high-glucose media. The growth curve was plotted by cell counting every 24 h for 7 days, using trypan blue due exclusion assay on the cells treated with verapamil (50 μM, red line) versus untreated cells (black line). Data are presented as mean ± SEM using one-way ANOVA with Tukey’s multiple comparisons test. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control.
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