Extended Data Fig. 2: Mitochondrial Ca²⁺ uptake in TMEM65-expressing cells.

a-b, No effect of TMEM65 depletion on mitochondrial Ca²⁺ uptake. Mitochondrial Ca²⁺ uptake was measured in digitonin-permeabilized WT, TMEM65-KD1, and TMEM65-KO1 HEK293 cells (the KO line was described in Fig. 5). Representative Ca²⁺ flux traces (a) and quantified uptake rates (b) demonstrate that TMEM65 depletion does not alter mitochondrial Ca²⁺ uptake. These results confirm that TMEM65 contributes specifically to mito-NCX without affecting other mitochondrial Ca²⁺ transport pathways. Uptake rates (AU/s) were normalized to the amplitude of fluorescence increase upon Ca²⁺ addition, yielding normalized Ca²⁺ uptake rates (s⁻¹) shown in panel b. Independent biological replicates: WT = 9; TMEM65-KD = 6; TMEM65-KO = 5. c, No effect of TMEM65 expression on mitochondrial Ca²⁺ uptake in Sf9 cells. Mitochondrial Ca²⁺ uptake was measured in permeabilized Sf9 cells with or without human TMEM65 expression. Representative Ca²⁺ flux traces (left) and quantified uptake rates (right) demonstrate that TMEM65 expression does not alter mitochondrial Ca²⁺ uptake. In contrast, hME expression enhances Ca²⁺ uptake, consistent with the uniporter serving as the primary pathway for Ca²⁺ entry into mitochondria. Independent biological replicates: No TMEM65 = 5; TMEM65 = 6; hME+TMEM65 = 6. d, No contribution of TMEM65 to Ca²⁺ influx. Digitonin-permeabilized Sf9 cells were treated with Ru360 to inhibit the uniporter. Following the addition of Ca²⁺, no mitochondrial Ca²⁺ uptake was observed regardless of TMEM65 expression. These results indicate that TMEM65 does not mediate Ca²⁺ influx into mitochondria. Each experiment, repeated 3 times independently, led to similar results. Data in this figure are presented as mean ± SEM. Statistical analysis was performed using unpaired, two-tailed t-test.

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