Figure 5.

Depletion of intracellular Ca²⁺stores and inhibition of mitochondrial Ca²⁺uptake in intact cells limits maximal mitochondrial respiration.A–C, AML12 cells were preincubated for 1 h in the absence (control) or presence of an intracellular Ca²⁺ chelator, BAPTA-AM, at different concentrations (5–20 μM, as shown), and O₂ consumption rates (OCRs) were measured using a Seahorse Extracellular Flux Analyzer. B, F = 1.683, p = 0.2469. C, F = 21.05, p = 0.0007, ∗∗∗p = 0.0008 versus CT. D–F, AML12 cells were incubated for 1 h in the absence (control, dimethyl sulfoxide [DMSO]) or presence of 10 μM of RuRed or CGP, and OCRs was measured using a Seahorse Extracellular Flux Analyzer. E, F = 0.5002, p = 0.5353. F, F = 3.799, p = 0.0692, ∗p = 0.0469 versus CT. G–J, MCU and NCLX silencing. G, NCLX mRNA levels after 48, 72, and 96 h of transfection with specific siRNA against NCLX (siNCLX) or a negative control (siCT), ∗p = 0.0332 (versus siCT 72 h) and ∗p = 0.0438 (versus siCT 96 h). H, MCU mRNA levels after 48 and 72 h of transfection with specific siRNA against MCU (siMCU) or a negative control (siCT). ∗p = 0.0209 (versus siCT 48 h) and ∗p = 0.0109 (versus siCT 72 h). I, representative Western blots of MCU and β-actin (internal control) after 72 h of transfection with specific siRNA against MCU (siMCU) or a negative control (siCT). J, densitometric analysis of the Western blots. ∗∗∗∗p < 0.0001 versus siCT. K–M, cytosolic Ca²⁺ levels in siMCU or siNCLX cells. K, typical cytosolic Ca²⁺ measurements performed using Fura-2AM. L, quantification under basal conditions. M, quantification of the peak after the addition of thapsigargin (TG). N–P, OCRs of siCT, siMCU, or siNCLX AML12 cells were measured using a Seahorse Extracellular Flux Analyzer. Oligomycin (oligo, 1 μM), CCCP (5 μM), and rotenone + antimycin (R/AA, 1 μM each) were added where indicated. B, E and O, quantification of basal respiration. C, F and P, quantification of maximal respiration. L, F= 4.206, p = 0.1172; M, F = 1.177, p = 0.3611; O, F = 1.348, p = 0.3308; P, F = 5.169, p = 0.0566, ∗p = 0.0446 versus siCT. Results are expressed as means ± SEM of 3 to 4 independent experiments and are expressed as representative traces (A, D, K and N) or means ± SEM of 3–5 independent biological experiments; Student’s t test or one-way ANOVA followed by Šidak.