Fig. 3

SCD1 expression is able to predict the response of BRAF-mutated-melanoma cells to targeted agents. a) Single-cell suspensions of melanoma cell lines were seeded onto a 96-plate ultra low attachment in sphere medium (3D) or in 96-plate cultured in RPMI-1640 (2D). Cell cultures treated with increasing concentrations of BRAF inhibitor (BRAFi) or MEK inhibitor (MEKi) (0.07–20 μM) alone or in simultaneous combination. After 7 days of treatment the sphere-forming efficiency (%) of 3D cancer cells was compared to untreated cells. In parallel the proliferation (%) of 2D cancer cells was compared to control (CTRL). Inset shows the evaluation of drug effects (IC50 value in 3D (red) and in 2D (blu)) performed on melanoma cell lines by Calcusyn software; b) Representative images of sphere formation of first generation taken on day 4. Scale bars: 50 μm. Single-cell suspensions of M14, A375, Mel 66 and M14-R cell lines were seeded in sphere medium and simultaneously treated with BRAF and MEK inhibitors alone or in combination for 4 days; c) Morphometric analysis of spheroids from treated or untreated M14 and A375. The median value plotted in boxplots showed that treated cultures were characterized by a higher size of spheroids compared to the untreated cultures; d) Immunofluorescence analyses on JARID1B expression were performed on M14, A375 and M14-R spheroids after 96 h of exposure to BRAF or MEK inhibitors and their combination; Scale bars: 50 μm; e) mRNA expression of jarid1b was determined by qRT-PCR after 96 h of drugs exposure. Jarid1b results upregulated by BRAF/MEK inhibitors on M14 and a375 cell lines. All data represent the means and SD of 3 independent experiments and are statistically significant if p < 0.05 (Anova test); f) Percentage of JARID1B positive cells treated with BRAF and MEK inhibitors and their combination; g) Stemness markers analysed on M14 and A375 cell lines after BRAF plus MEK inhibitors by qRT-PCR