Analysis of NADH autofluorescence. A: Mitochondrial and nuclear NADH autofluorescence levels (mean ± SEM of a minimum of 3 independent experiments) expressed in absolute grey levels. Note that the signals were obtained from cells with clearly distinct morphology, which makes intercomparison of Prim-MEF, Imm-MEF and TBX2-MEF on the one hand, and Ras-transformed populations on the other, difficult. Absolute NADH autofluorescence increases gradually with passage number in H-RasV12/E1A-transformed cells. B: Representative recordings of NADH autofluorescence in Ras-LP and Ras-TUM cells before and after rotenone application. Rotenone inhibition of Complex I results in a differential increase in NADH autofluorescence. The percentage NADH autofluorescence is indicative of different rates of mitochondrial respiration. These data confirm the relatively higher respiration rates in Ras-LP cells. C: Cumulative data from n = 3 NADH autofluorescence/rotenone analyses carried out on separate days, with a minimum of 3 coverslips per day. Note that this parameter is cell morphology-independent. **:p < 0.01 compared to Prim-MEF ##:p < 0.01 for Ras-LP – Ras-TUM intercomparison (one-way ANOVA/Bonferroni). D: Superoxide levels expressed as fluorescence signal of HEt oxidation products per cell. Ras-LP levels were arbitrarily set at 100%. Oxygen consumption data per cell are available in Additional file 1, Fig. S4B and Fig. S4D. HEt fluorescence in Ras-LP, HP and TUM cells was determined in three independent experiments, each in duplicate, with at least 20,000 cells per assay. Prim-MEF, Imm-MEF and TBX2-MEF HEt fluorescence was determined in one experiment, in duplicate. Superoxide levels correlate with respiration rates measured in (B) and (C). ##:p < 0.01 compared to Ras-LP in a Student's t-test.