TMX1 regulates mitochondrial energy production. (A) Measurement of the mitochondrial oxygen consumption rate, expressed as the maximal respiration ETC capacity (picomoles of O₂ per second per citrate synthase (CS) activity, see Materials and methods) in control HeLa cells and TMX1 heterozygous KO HeLa cells. Results from three independent experiments are summarized as a graph (*, P = 0.026; standard error indicated). (B) Measurement of the mitochondrial oxygen consumption rate (picomoles O₂ per second per CS activity, see Materials and methods) in control HeLa cells and cells transfected with siTMX1. Results from three independent experiments are summarized as a graph (*, P = 0.014; standard error indicated). (C) Measurement of the mitochondrial oxygen consumption rate (picomoles O₂ per second per CS activity) in control A375P cells and cells transfected with FLAG-tagged TMX1 or siTMX1. Results from three independent experiments are summarized as a graph (*, P = 0.018; standard error indicated). (D) Measurement of cellular ATP content (see Materials and methods) in control HeLa and heterozygous TMX1 KO HeLa cells. Results from three independent experiments are summarized as a graph (n = 9; **, P = 0.042; standard error indicated). (E) Measurement of cellular ATP content in control HeLa and heterozygous TMX1 KO HeLa cells treated with 10 µM BAPTA-AM and 10 mM NAC. Results from three experiments are summarized. **, P = 0.001 for WT BAPTA-AM, 0.05 for KO BAPTA-AM; **, P = 0.001 for WT NAC, 0.12 for KO NAC; standard error expressed versus HeLa. (F) Analysis of HeLa control and TMX1 KO and A375P lysates transiently transfected as indicated with RNAi against TMX1. Lysates were probed for phospho-AMPK and normalized to total AMPK. Representative gels are shown. The graph plots the levels of phosphorylated AMPK in cells transfected with TMX1 RNAi versus control cells transfected with scrambled RNAi (n = 3; **, P = 0.003 for HeLa; *, P = 0.02 for A375P; standard error expressed vs. scrambled controls).