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Figure 3 | Journal of Translational Medicine

Figure 3

From: Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

Figure 3

Anti-ErbB3 mAb A4 counteracts the increase of ErbB3-dependent AKT phosphorylation and potentiate growth inhibition induced by GSK1120212b. (a) LOX IMVI melanoma cells were serum starved and treated with vemurafenib (0.3 μM), with GSK1120212b (GSK, 0.15 μM) or with their combination in presence or not of anti-ErbB3 mAb A4 (20 μg/ml) for 24 h. Western blot analysis shows that A4 mAb abrogate ErbB3 phosphotylation as well as the strong increase of pAKT induced by both inhibitors. 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. (b) Cells were grown in the presence of different doses of GSK combinated or not with A4 mAb (20 μg/ml) for 10 day. Cells were then dissolved in a Methanol/SDS solution and the adsorbance (595 nm) was read as above. Quantitative analysis for curve fitting and for IC50 evaluation, performed as above, shows that the treatment with A4 enhances the inhibitory effect of GSK on cell growth (IC50 GSK = 115 nM; IC50 GSK + A4 = 19 nM). p-values were calculated and significance level has been defined as above. For IC50 GSK + A4 p < 0,001 vs IC50 GSK. (c) Cells were treated with suboptimal doses of vemurafenib, GSK or their combination in presence or not of A4 mAb (c). The in vitro colony formation assay shows that the addition of A4 significantly inhibits cells growth. *p < 0,01 vs vem-treated or GSK-treated cells; ** p < 0,001 vs vem + GSK- treated cells; NS vs untreated cells.

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