Figure 2

Anti-ErbB3 mAbs differently counteract the increase of ErbB3-dependent AKT phosphorylation and potentiate growth inhibition induced by vemurafenib. LOX IMVI melanoma cells serum starved and treated with vemurafenib (0.3 μM) for 24 h were incubated or not with 20 μg/ml of anti-ErbB3 mAbs A4 (a), A3 or A2 (d). Western blot analysis shows that A4 and A3, but not A2 mAbs abrogate receptor phosphorylation and ATK signaling. 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. LOX IMVI cells were grown in the presence of different doses of vemurafenib alone or in combination with 20 μg/ml of A4 (b), A3 or A2 (e) mAbs for 10 days. Cells were then fixed and stained with crystal violet (c). Cells were then dissolved in a Methanol/SDS solution and the adsorbance (595 nm) was read using a microplate ELISA reader (b, e). Quantitative analysis for curve fitting and for IC50 evaluation, performed by KaleidaGraph software, shows that the treatment with A4 and A3 but not with A2 enhances the inhibitory effect of vemurafenib on cell growth (IC50 vem = 155 nM; IC50 vem + A4 = 36 nM; IC50 vem + A3 = 62, IC50 vem + A2 = 146 nM). Results are reported as mean values ± standard deviation (SD) from three independent experiments. p-values were calculated using Student’s t test and significance level has been defined as p < 0,05. For IC50 vem + A4 and IC50 vem + A3 p < 0,001 vs IC50 vem; IC50 vem + A2 NS vs IC50 vem.