Fig 2: Recombinant IL-6-induced p38-MAPK activation mediates elevated WNT5A expression and increased melanoma cell invasion. (A) Western blot analysis of p38-MAPK phosphorylation (p-p38) in the human melanoma cell lines WM852, HTB63, A375 and A2058 treated with Carrier (0.1% BSA in PBS) alone or recombinant IL-6 (20 ng/ml) for 60 min. Densitometric analyses were performed, and the data were then normalized against total p38-MAPK (p38; n = 3). (B) Western blot analysis of p38-MAPK phosphorylation (p-p38) in HTB63 cells incubated in the absence or presence of recombinant IL-6 (20 ng/ml) for 30 min, 1 h, 2 h, 3 h or 4 h. Densitometric analyses were performed, and the data were then normalized against total p38-MAPK (p38; n = 3). (C–D) Western blot analysis of WNT5A expression in HTB63 and A375 cells treated with either Carrier (0.1% BSA in PBS) alone or recombinant IL-6 (20 ng/ml) for 24 h in the absence (only DMSO) or presence of SB203580 (10 µM dissolved in DMSO). Densitometric analyses were performed, and the data were then normalized against a-tubulin (n = 4). (E) Matrigel invasion of human HTB63 melanoma cells treated with recombinant IL-6 (20 ng/ml, dissolved in 0.1% BSA in PBS) in the absence or presence of SB203580 (10 µM dissolved in DMSO) and recombinant WNT5A (rW5A, 0.2 µg/ml, dissolved in 0.1 mM EDTA, 0.5% CHAPS, 0.1% BSA in PBS) or their respective controls, Carrier (0.1% BSA in PBS), DMSO and Vehicle (0.1 mM EDTA, 0.5% CHAPS, 0.1% BSA in PBS). The invasion assays were performed over 24 h (n = 4) and analyzed as described in Materials and methods. The results are given as means and S.E.M; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.