Fig. 1: Mutant K-Ras overexpression induces cell transformation.

A Western blot analysis of protein extracts from cells overexpressing Vector, WT, GV, or GD K-Ras cultured for 48 h. The overexpressed proteins WT K-Ras and mutant K-Ras GV and GD are V5-tagged. V5 tag, phosphoERK1/2, and ß-actin were used as primary antibodies. B Observation of morphology of cells overexpressing Vector, WT, GV, or GD K-Ras after 48 h in culture using the bright field. Cell morphology varies across different conditions. C Cell growth analysis using quantification of the cell number over the course of 5 days in culture. Cells overexpressing Vect, WT, GV, or GD K-Ras were plated in a growth medium that contains EGF. The next day, called day 0, the medium was changed to either growth medium (full medium, in black) or growth medium where EGF was not added (EGF-free medium, in gray). Cells were trypsinized and number of viable cells was determined using a hemocytometer after 1, 3, and 5 days (N = 4, data presented as mean ± SEM, two-way ANOVA with Sidak’s multiple comparisons test, * means p < 0.05 comparing the group full medium to the group EGF-free medium at the same time point). D Colony formation assay using cells overexpressing Vect, WT, GV, or GD K-Ras. Cells were plated and cultured in soft agar for 2 weeks and then finally stained overnight using a membrane-permeable dye that stains viable cells. E Quantification of the number of colonies obtained after culturing cells overexpressing Vect, WT, GV. or GD K-Ras for 2 weeks in soft agar (N = 4, data presented as mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, * means p < 0,05 comparing the tested groups to the group control Vector).