Lactic acid production, oxygen consumption and proton production rate. A: Lactic acid production in μ moles/mg protein (mean ± SEM). Lactic acid production was measured in media samples taken from the cell cultures used to obtain the cell doubling time data in Table 1. Six independent experiments, each in duplicate, were conducted for all cell populations, except Ras-HP (n = 8) and Ras-TUM (n = 7). Lactic acid production increases with passage number in H-RasV12/E1A-transformed cells. ***:p < 0.001 compared to Prim-MEF. #:p < 0.05; ##:p < 0.01; ###:p < 0.001 for Ras-LP – Ras-HP – Ras-TUM intercomparisons (one-way ANOVA/Bonferroni). B: Oxygen consumption in NRFU/min/mg protein measured in BD oxygen biosensor plates (mean ± SEM). Each experimental value represents n = 3 (Prim-MEF, Imm-MEF, TBX2-MEF) or n = 6 (other cell populations) independent assays, each carried out in triplicate. Cells were seeded at 200,000 cells per well of a 384-well plate. Oxygen consumption peaks immediately after H-RasV12/E1A transformation, and then decreases with passage number. ***:p < 0.001 compared to Prim-MEF. ###:p < 0.001 for Ras-LP – Ras-HP – Ras-TUM intercomparisons (one-way ANOVA/Bonferroni). C: Proton production rate (PPR) and oxygen consumption rate (OCR) per cell measured in Seahorse XF24 analysis 48 hours post seeding (mean ± SEM; n = 6, except Ras-HP, n = 4). Prim-MEF, Imm-MEF and TBX2-MEF cells were seeded at 25,000/well. Ras-LP, Ras-HP, Ras-TUM cells were seeded at 30,000/well. PPR: **:p < 0.01 compared to Prim-MEF. OCR: ##:p < 0.01 compared to Prim-MEF (one-way ANOVA/Bonferroni). These data confirm the gradual increase in cellular acidification and decrease in oxygen consumption described in (A) and (B) in real time analysis.