Figure 1.

Insulin does not influence proliferation but markedly affects the subpopulation of NGFR-positive cells and induces AKT activity in melanoma cells, either transiently or sustainably. (A) Proliferation curves of melanoma cells grown in the presence (black line) or absence (orange line) of insulin based on the activity of acidic phosphatase (APA assay). n=3. (B and C) The percentages of Ki-67-positive cells (B) or NGFR-positive cells (C) in the populations of melanoma cells grown without insulin relative to their percentages in the insulin-treated melanoma cell populations, 42 h after cell seeding. Data are presented as mean values of three biological replicates ± SD. Student’s t-test was used to calculate statistical significance. Differences are considered significant at *P<0.05. (D) Western blot analysis of the activity of PI3K/AKT and MAPK pathways in melanoma cells grown in the presence or absence of insulin for 24 h, shown as levels of phosphorylated AKT at Ser473 (pAKT^S473) and Thr308 (pAKT^T308), and phosphorylated ERK1/2 (pERK1/2), respectively. n=2. (E) The level of phosphorylated AKT at indicated time points following insulin stimulation, shown as representative Western blots. Total AKT and GAPDH served as loading controls. (F) Densitometry was used to analyze band intensity. The level of phosphorylated AKT corrected by total AKT level measured at the indicated time points was expressed relative to corrected pAKT level at 3 h of insulin treatment. Data from two independent experiments are expressed as mean ± SD.