Fig. 5

R-RAS2 is functionally downstream of CD98/LAT1 controlling mTORC1 activation and breast cancer cell metabolism. a, Seahorse analysis of the oxygen consumption rate (OCR) of WT and RRAS2 knockdown CBM-MBC21 cells in complete medium. Measurements were made in sextuplicate after plating the two types of cell at different densities (15,000 cells/well and 20,000 cells/well). The drugs (oligomycin, FCCP and rotenone + antimycin A) were added sequentially to measure different respiratory parameters. Datapoints show the mean ± s.e.m. b, Respiratory parameters calculated from the Seahorse data (Fig. 5a), the significance of which was assessed with a two-way ANOVA and Sidak’s multiple comparison test. **, p = 0.004; ***, p = 0.0009. c, Western blot analysis was performed to assess signaling pathway activity by examining the phosphorylation of key residues in the indicated elements. Control and CBM-MBC21 knockdown cells were deprived of the amino acids Leu, Ile, Val, and Gln for 1.5 h, followed by replenishment of the specified amino acids for the indicated time points (in minutes). Post-nuclear cell lysates were analyzed via Western blotting. As controls, lysates from cells maintained in full medium throughout the experiment and from cells treated with 50 nM rapamycin in full medium were included. d, Quantification by densitometry of Western blots as in Fig. 5c run in triplicate. Bar plots show the mean ± s.e.m of densitometry data referring the intensity of the phosphoprotein bands to that of total Akt. Significance was assessed using a two-way ANOVA test. **, p = 0.002; ***, p = 0.0002; ****, p < 0.0001. e, Effect of amino acids Leu, Ile, Val and Gln deprivation followed by replenishment on the phosphorylation of S6 protein at Ser240 analyzed by flow cytometry. Datapoints represent the mean ± s.e.m of triplicates. Significance was assessed using a two-way ANOVA Sidak’s multiple comparison test