Fig. 2

Mitochondrial mass and activity in tumor cells. A Left: MitoTracker Red staining of normal human renal epithelial cells (HRE) or A-498, SNC-12 and 786–0 clear cell carcinoma cells (Scale bars = 10 μm). Right: Relative fluorescence intensity (RFI) of MitoTracker in single cell along the diameter. B Representative FACS profile of mitochondrial mass labeled with MitoTracker in SNC-12, 786–0 and A-498 cells, reported as median of fluorescence intensity (MFI). CRT indicates the unstained control of each cell line. C Graphical representation of mean fluorescence intensity (MFI) measured by FACS. D Oxygen consuming rate (OCR) by mitochondrial stress test (MST using Seahorse system) in A-498, SNC-12 and 786–0 cells plated in 12 × multiwell plates at 1 × 10.⁵ cells/well. Data are reported per µg of protein under basal conditions and in response to mitochondrial inhibitors (oligomycin; FCCP; rotenone). E Quantification of basal respiration, ATP production, proton leak and maximal respiration in tumor cells. Basal respiration is the value just before oligomycin injection, minimal respiration is the lowest value after oligomycin injection, and maximal respiration is the highest value after FCCP injection. All values were calculated after subtraction of non-mitochondrial respiration. F Extracellular acidification rate (ECAR) reported as milli pH per minute (mpH/min) under basal conditions in A-498, SNC-12 and 786–0 cells over the time. G ECAR in milli pH per minute (mpH/min)/ µg under basal conditions over the time vs. OCR under conditions of basal respiration in A-498, SNC-12, 786–0 and HRE cells. Abbreviations: Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone; Rotenone. Data are mean ± SEM. of three independent experiments in triplicated. P-values were calculated by Mann–Whitney U or Student's t-tests. **p < 0.01