Figure 1
From: Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells
Expression and functional effects of allotopic nND1. (A) Western blot analysis for ND1 in control (CC), OS-93 and OS-93ND1 representative clones. Voltage-dependent anion channel (VDAC) was used as a loading control. One representative experiment of three is shown. (B) m.3571insC mutation load evaluation by fluorescent (F)-PCR. Wild-type and mutant fragments are distinguished based on the length of the homopolymeric stretch, where 7C corresponds to the C insertion. (C) Complex I in-gel activity (CI-IGA) assay in isolated mitochondria. CI-IGA band is indicated with an arrow. One representative experiment of four is shown. (D-E) NAD+/NADH ratio and NADH levels were measured in cell lysates. Data (mean ± SD) are expressed as pmoles of NADH and normalized for protein content (n = 3; *P <0.05; **P <0.01). (F) Oxygen consumption rate (OCR). Measurements were performed upon injection of 1 μM oligomycin (O), 0.1 μM trifluorocarbonylcyanide phenylhydrazone (FCCP) (F), 1 μM rotenone (R) and 1 μM antimycin A (AA). The inset shows the OCR control cell line (CC) profile, analogous to that of OS-93ND1 cells. Data (mean ± standard error of the mean (SEM)) are expressed as pmoles of O2 per minute per 3 × 104 cells (n = 3). (G) Mitochondrial ATP synthesis driven by pyruvate/malate and succinate, CI and CII substrates, respectively. CS, citrate synthase. (n = 4; #undetectable value; **OS-93 vs CC, P <0.01; **OS-93ND1 vs OS-93, P <0.01; *CC vs OS-93ND1, P <0.05). (H) Mitochondrial membrane potential evaluation in CC, OS-93 and OS-93ND1 cells. Arrows indicate the addition of 6 μM oligomycin (O) and 4 μM FCCP (F). Data are mean ± SEM (n = 6). Fluorescence readings following the addition of oligomycin and preceding that of FCCP revealed a statistically significant difference (P <0.05) for all time points between OS-93 and both CC and OS-93ND1 cells.