Figure 3.

Insulin protects melanoma cells against vemurafenib- or trametinib-induced apoptosis and diminishes drug-triggered downregulation of Ki-67 expression. (A) Western blot analysis of the effects of 10 µM vemurafenib (PLX) or 50 nM trametinib (TRA) on the activity of MAPK (level of pERK1/2) and PI3K/AKT (level of pAKT^S473) pathways, and PARP cleavage in the presence or absence of insulin, after 24 h of drug treatment. GAPDH was used as a loading control. n=3. (B) Insulin-induced changes in the percentage of Ki-67-positive melanoma cells, untreated or treated with PLX or TRA for 40 h. n=3. (C) Apoptosis shown as percentages of Annexin V-positive cells assessed by flow cytometry after 30 and 46 h of treatment with PLX or TRA, in the presence or absence of insulin. Bars represent mean values of three biological replicates ± SD. Statistical significance marked as asterisks in proximity to each bar refers to the drug-induced difference in relation to control, and as asterisks adjacent to lines above the bars refers to the difference between melanoma cells grown with or without insulin. Statistical significance for both panels was calculated with ANOVA followed by Scheffé’s test. Differences are considered significant at *P<0.05.