Fig. 3.

Apoptosis/necrosis induced by MET/6-AN treatment. Melanoma cells were treated with metformin (MET, 5 mM), 6-aminonicotinamide (6-AN, 50 μM) or a combination of both (MET/6-AN). (A) After 72 h of treatments, non-fixed cells were incubated with acridine orange/ethidium bromide (AO/EB) to be evaluated by epifluorescence microscopy. The arrows on the representative images of AO/EB staining indicate the co-localization of both stains showing late apoptotic or necrotic events (orange/red). (B) After 48 h treatment, Annexin V antibody and propidium iodide (PI, 5 μg/mL) label was detected by flow cytometry as described in M&M. Results are expressed as dot plot graph and stacked bar chart with means of three independent experiments. (C) After 48 h treatment, the SubG0 population was determined. Results are expressed as means ± SEM of two independent experiments. Statistical analysis was performed with one-way ANOVA using Tukey's multiple comparisons test to calculate significance (*p < .05 and ***p < .001 respect to control treated with PBS, ○○○p < .001 respect to MET 5 mM and ●●●p < .001 respect to 6-AN 50 μM). (D) Representative image of Poly (ADP-ribose) polymerase-1 (PARP1) expression after 48 h of treatments. Values refer to the relative density of cleaved PARP with the respective β-actin. Results are expressed as means ± SEM of two independent experiments.