Figure 4

Vemurafenib treatment induces increased expression and release of neuregulin (HRG) in LOX IMVI melanoma cells. Cells were serum starved for 24 h and treated or not with vemurafenib (0,3 μM) for 1, 6 or 24 h. Real-time PCR analysis performed using specific TaqMan probes (a) and Western blot analysis performed using anti-HRG antibody (b) show that upon 24 h of vemurafenib treatment HRG expression is increased at both mRNA and protein levels. For PCR and Western Blot analyses results are reported as mean values ± standard deviation (SD) from three independent experiments. (c) Cells were treated with vemurafenib in presence or not of a neutralizing anti-HRG antibody. The Anti-ErbB3 A2 mAb was used as negative control. The anti-HRG antibody, but not A2 mAb, strongly inhibits both ErbB3 and AKT phosphorylation triggered by vemurafenib. (d) Cells treated for 1 h with the conditioned medium (CM) from untreated-LOX IMVI cells (untreated CM) or from vemurafenib-stimulated LOX IMVI cells (vem CM) were pre-incubated or not with the anti-HRG antibody or with A2 mAb. The treatment with the anti-HRG antibody, but not A2 mAb abrogates both ErbB3 and AKT phosphorylation induced by vem CM. For densitometric analysis pErbB3/ErbB3, pERK/ERK and pAKT/ATK values are expressed as fold change with respect to the control unstimulated cells to which value = 1 was assigned. Results are expressed as mean values from three independent experiments.