Decreased expression of TMX1 inhibits ER-derived Ca²⁺ flux. (A) Measurement of ER Ca²⁺ content change after histamine-mediated ER calcium release. HeLa control and heterozygous KO cells were transfected with LAR-ER-GECO. Ca²⁺ was released with histamine, and probe fluorescence was recorded before and after 50 µM histamine treatment by fluorescence microscopy. Relative fluorescence decrease was plotted and quantified from three independent experiments (P = 0.017; standard error expressed vs. HeLa). (B) Measurement of cytosolic Ca²⁺ concentration after histamine-mediated ER Ca²⁺ release. Representative FURA-2-derived curves from TMX1 heterozygous KO and KD cells are plotted and quantified from five independent experiments; standard error expressed versus HeLa or scrambled control (*, P = 0.02 for WT vs. KO). (C) Measurement of cytosolic Ca²⁺ clearance after histamine-mediated ER calcium release. HeLa control and heterozygous KO cells were loaded with FURA-2. Ca²⁺ was released with histamine and probe fluorescence was recorded before and after 50 µM histamine treatment on a fluorimeter. Data were derived from four independent experiments (*, P = 0.014; standard error expressed vs. HeLa). (D) Measurement of mitochondrial Ca²⁺ after histamine-mediated ER Ca²⁺ release. HeLa and TMX1 heterozygous KO HeLa cells were transfected with mitochondrial R-GECO1 and treated with 50 µM histamine, followed by probe fluorescence recording. Results are summarized as a graph representing maximum response. Data were derived from six independent experiments for with Ca²⁺ (*, P = 0.01) and from three independent experiments for without Ca²⁺; standard error expressed versus HeLa. (E) Measurement of mitochondrial Ca²⁺ after histamine-mediated ER Ca²⁺ release. HeLa and A375P cells transfected with shTMX1 plasmids and mitochondrial R-GECO1 were treated with 50 µM histamine, and probe fluorescence was recorded. Results from three independent experiments each are summarized as a graph representing maximum response. **, P = 0.001 for with Ca²⁺; *, P = 0.05 for without Ca²⁺. (F) Electron micrograph analysis of control and TMX1 heterozygous KO HeLa cells. One representative electron micrograph, respectively, is shown. Analysis and quantification of the distance of 595 MAMs for WT and 648 MAMs for KO is shown as a bar graph; standard error indicated (***, P = 0.0001). Bars, 150 nm.