Figure 4. Long-term RSK inhibition significantly impairs growth of resistant melanoma cells.

(A, B) Clonogenic assay of MAPK inhibitor resistant cells after a 10 d-treatment with signalling pathway inhibitors. Cultures were stained with Coomassie Brilliant Blue. Images are representative of three independent experiments. Vemurafenib resistant SKMel28 R were treated with increasing concentrations of RSK inhibitor or vemurafenib, either administered alone or in combination with a fixed concentration of RSK inhibitor (left panel: BI-D1870; right panel: LJH-685) (A). Double resistant SKMel28 RR were treated with ascending concentrations of vemurafenib, trametinib and BI-D1870, either alone or in combinations (B). (C–E) Anchorage-independent growth assays of MAPK inhibitor resistant cells treated with signalling pathway inhibitors for 10 d. Colonies were visualized with crystal violet, counted and normalized to the untreated control. Representative data of two independent experiments is shown (mean ± SD, n = 3). Significance was determined by one-way ANOVA with subsequent Tukey's multiple comparisons test. Single (left panel) and double resistant (right panel) SKMel28 cells were treated with vemurafenib, the RSK inhibitor BI-D1870 or the combination (C). In (D), double resistant SKMel28 were treated either with the combination of vemurafenib and trametinib, with BI-D1870 or the triple combination. In (E), single (left panel) and double resistant (right panel) SKMel28 cells were treated with vemurafenib, the RSK inhibitor LJH-685 or the combination. (F) Organotypic skin reconstructs with SKMel28 R melanoma cells treated with vemurafenib (5 μM), either alone or in combination with BI-D1870 (5 μM) for 10 days. Sections were stained with hematoxylin and eosin or with Ki67-specific antibodies. Two representative images per treatment are shown, respectively (n = 3). The scale bar indicates 100 μm.