(A) Bicistronic vectors were used to transduce BMEL cells that were sorted by flow cytometry according to fluorescence intensities, giving rise to RasLOW and RasHIGH populations labelled with either Venus or mCherry. RasG12V, Venus and mCherry expression were quantified by RTqPCR (see also Figure 1—figure supplement 1), and results for cell lines BMELWT, RasLOW-Cherry, RasHIGH-Venus are shown as mean ± SEM. p-Value of unpaired t-test statistical test is shown. Differences in RasG12V expression were confirmed by western blot analysis (RasLOW-cherry versus RasHIGH-Venus). Actin was used as loading control. (B) Parental non-transformed BMEL (‘BMELWT’), RasLOW and RasHIGH cells were seeded in soft agar, stained with crystal violet and counted after 21 days of culture. Representative wells and mean colony numbers ± SEM from two independent experiments performed in triplicate are shown. (C) Phosphoprotein array performed on BMELWT, RasLOW, and RasHIGH cell lines. Bottom panel shows the quantification of duplicates from two independent experiments. a.u.: arbitrary units. (D) qPCR quantification of Erk target genes signature in RasLOW and RasHIGH BMEL cells normalized to expression in parental BMELWT cells. Mean and SEM from three to four independent experiments are shown. Statistical significance from unpaired t-test are indicated. (E) Venn diagram and heatmaps showing log2 RPKM values of genes regulated by RasG12V in BMEL cells detected by an RNAseq transcriptomic analysis (log2FC >1.5, p-value <0.01). Genes expressed at low levels (all RPKM log2 values <1) were removed from the analysis. From left to right: 160 genes were modulated by Ras similarly in RasLOW and RasHIGH populations, 65 genes were altered only in the RasHIGH cells and the expression of 15 genes was gradually modulated in the RasLOW and RasHIGH populations. (F) Volcano plot presentation of deregulated genes in RasHIGH cells versus parental BMEL cells. Genes belonging to regulation of immune response signature (GO:0050776) are highlighted in orange. ns: not significant, *<0.05, **<0.01, ***<0.001, ****<0.0001.