Figure 4. GH-excess promotes and GHRKD attenuates markers of epithelial mesenchymal transition (EMT) in human melanoma cells.

a-c. Relative RNA expression was quantified for N-cadherin (a), vimentin (b) and E-cadherin (c) in SK-MEL-28 melanoma cells following addition of 5, 50 and 150 ng/mL hGH or 24 hr following GHRKD, in presence or absence of 0 and 50 ng/mL hGH. Similar results for MALME-3M, MDA-MB-435 and SK-MEL-5 human melanoma cells are presented in Supplementary Figure 4. In all cases, RNA expressions were normalized against β-actin and GAPDH values as reference genes and compared against untreated control. [*, p < 0.05, Wilcoxon sign rank test, n = 4] d-f. Densitometry analyses of relative protein expressions of N-cadherin (d), vimentin (e), and E-cadherin (f) as estimated by western blot (WB) of lysates of SK-MEL-28, MALME-3M, MDA-MB-435 and SK-MEL-5 cells, collected 60 hr post-transfection with either scramble (scr)-siRNA or GHR-siRNA in presence of GH. g. Representative images of WB analyses of phosphorylation levels of N-cadherin, vimentin and E-cadherin in four melanoma cell lines. WB was performed using appropriate antibodies. Densitometry analyses of individual blots was performed using ImageJ software and the ratio of phosphorylated vs. total protein levels against untreated scr-siRNA transfected controls. Overall, excess GH promoted while GHRKD reversed EMT in human melanoma cells. Blots from individual experiments were quantified and the mean of three blots per antibody was taken. Protein levels were normalized against expression of β-actin. [*, p < 0.05, Students t test, n = 3].