Fig 2: RT-PCR of Pyk2, CREB, and MCU demonstrates that these mRNAs are reduced in TRPM2-depleted cells. mRNA was prepared from scrambled control and TRPM2-depleted SH-SY5Y cells which were treated with doxorubicin for 48 h. RT-PCR was utilized to measure mRNA expression. Relative amounts were calculated compared with scrambled. *P < 0.05, statistically reduced expression of mRNA in the KO compared with scrambled analyzed in triplicate in three experiments (Student’s t-test). CREB, cAMP-responsive element-binding protein; KO, knockout; Pyk2, proline-rich tyrosine kinase 2; TRPM2, transient receptor potential melastatin channel subfamily member 2.

Fig 3: Genetic interaction between MCU and Complex I in Drosophila.a, c, e Drosophila survival for the indicated genotypes and sex. b, d, f Quantification of dwell time in the island assay for indicated genotypes for female flies. a, b The muscle specific MHC-GAL4 was used to drive expression of NDUFB10RNAi, MCUWT, or MCU?NTD in wild-type or MCU1/MCU1 mutant flies, as indicated. c, d As in (a, b), except the dominant-negative pore mutant MCUDQEQ was expressed with MHC-GAL4. e, f As in (a, b), except the isolated NTD fragment was expressed with MHC-GAL4. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics: (a, c, e) Fischer’s Exact Test with Bonferroni correction; (b, d, f) Log-rank (Mantel-Cox) test with Bonferroni correction. Source data are provided as a Source Data file.

Fig 4: Diminished MCU degradation is responsible for enhanced uniporter activity in Tfam KO hearts, and prolongs survival.a Immunoblotting for the specified proteins in P10-P14 mouse heart lysates. Samples from 3 mice shown per genotype. b Analysis of Tfam and Mcu transcripts in mouse hearts. c Ca2+ uptake in isolated cardiac mitochondria incubated in Oregon Green BAPTA 6F (OGB6F). Arrow indicates 10 µM Ca2+ pulse. d Summary of the normalized rate of Ca2+ uptake after a 10 µM Ca2+ pulse. e Heart weight to body weight ratios of P10-P14 mice of the indicated genotypes. f Kaplan–Meier survival analysis of the Tfam KO mice compared to Tfam-Mcu DKO mice. Comparison via a log rank test. Summary data are presented as mean values ± SEM. Statistics: (b) Kruskal–Wallis followed by Dunn’s test with Bonferroni correction; (d, e) 1-way ANOVA followed by Bonferroni-corrected means comparisons; (f) Log-rank test. Source data are provided as a Source Data file.

Fig 5: Changes in the protein expression of distinct Ca2+ transport and binding proteins in PL and AM muscles during different periods. Histograms depicting (A) representative Western blot images of STIM1, ORAI1, RyR1, LETM1, PMCA3, SERCA1, MCU, MICU1, and CALM in PL and AM muscles during different periods. Histograms depicting (B) STIM1 protein expression, (C) ORAI1 protein expression, (D) RyR1 protein expression, (E) LETM1 protein expression, (F) PMCA3 protein expression, (G) SERCA1 protein expression, (H) MCU protein expression, (I) MICU1 protein expression, and (J) CALM protein expression in PL and AM muscles during different periods. PL, plantaris; AM, adductor magnus. SA, summer active group; PRE, pre-hibernation group; LT, late torpor group; IBA, inter-bout arousal group; ET, early torpor group; POST, post-hibernation group. Values are means ± SEM, n = 6–8. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with SA; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with PRE; & p < 0.05 compared with LT; $ p < 0.05 and $$ p < 0.01 compared with IBA; ++ p < 0.01 compared with ET.