Supplementary Figure S1.

Generation and validation of TIME transgenic cell lines stably expressing GNAQ^WT, GNAQ^R183Q, GNA11^WT, or GNA11^R183C alleles. (a) Schematic representation of the lentiviral expression vectors used to infect the TIME cell line and generate stable recombinant derivatives. (b) Sanger sequencing performed on TIME recombinant models using primers annealing to exon 4 of GNAQ or GNA11 genes. Chromatogram of GNAQ and GNA11 codon-183 for mutation confirmation. (c) Western blot analysis of TIME parental cells and TIME transduced with GNAQ^WT, GNAQ^R183Q, GNA11^WT, or GNA11^R183C lentiviruses. The cell lysates were probed with the indicated antibodies. (d) Western blot analysis of TIME parental cells and TIME transduced with GNAQ^WT or GNAQ^R183Q lentiviruses. The cell lysates were probed with the indicated antibodies. Quantification of the bands reported in figure represents the average of four independent western blot experiments. (e) Western blot analysis of TIME parental cells and TIME transduced with GNA11^WT or GNA11^R183C lentiviruses. The cell lysates were probed with the indicated antibodies. Quantification of the bands reported in figure represents the average of four independent western blot experiments. ERK, extracellular signal–regulated kinase; HA, hemagglutinin; HEK, human embryonic kidney; mut, mutated; pERK, phosphorylated ERK; TIME, telomerase-immortalised microvascular endothelial; WT, wild-type.