Fig. 3

RSK inhibitors effectively impair melanoma cell growth under three-dimensional growth conditions and in vivo. A, B Growth of melanoma spheroids embedded in soft agar under RSK inhibition. Microphotographs of the spheroids were taken over the course of treatment (A: right panel; scale bar represents 200 µm). Spheroid sizes were quantified with ImageJ and normalized to the initial spheroid size at treatment start (mean ± standard error of the mean (SEM); A: left panel; B). Two independent experiments with n ≥ 3 were performed. C, D Anchorage-independent growth assay of melanoma cell lines (C) and patient-derived short-term cultures (D) under RSK inhibitor treatment (PMD-026). After 10 d, colonies were visualized with crystal violet, counted, and normalized to the solvent-treated controls (N = 2 with n = 5, mean ± SD). Significance was determined by one-way ANOVA with subsequent Tukey’s multiple comparisons test. E, F Subcutaneous xenograft growth of melanoma cells (E: BRAF^Mut; F: NF-1^LOF) in NOD scid gamma (NSG) mice under RSK inhibitor therapy. BRAF-mutated 451LU were additionally treated with the BRAF^V600E/K inhibitor vemurafenib and its combination with PMD-026 (E). Mean tumor growth (± SEM; left panel) and tumor end volumes (violin plot including individual values, quartiles and median; right panel) for the respective treatment groups are shown (E: n_Vehicle, n_PMD-026 = 7, n_Vemurafenib, n_{PMD-026+Vemurafenib} = 6; F: n = 7). Significance was determined by one-way ANOVA with subsequent Tukey’s multiple comparisons test (E) or with a two-tailed unpaired t-test (F)