Fig 2: The SLC25A3 L175A variant restores mitochondrial Cu levels and rescues the cytochrome c oxidase (COX) deficiency in KO MEFs.(A) Immunoblot analysis of SLC25A3 abundance in Slc25a3 KO MEFs alone or those transduced with wild-type SLC25A3 (WT), a H75A variant (H75), or a L175A variant (L175). Actin served as an internal loading control. (B) COX activity in KO MEFs alone (n = 6) or transduced with WT SLC25A3 (n = 4), a H75A variant (H75) (n = 5), or a L175A variant (L175) (n = 6). ns, p>0.05, ****, p<0.0001 based on a one-way ANOVA. (C) Immunoblot analysis of SLC25A3, TOM40, and COX1 abundance in whole cells (WC) or isolated mitochondrial (M) from WT or KO MEFs alone (NA) or transduced with the SLC25A3 L175A variant (L175). (D) Total Cu levels in mitochondria from WT or KO cells as in (C), determined by ICP-OES. (E) Cu uptake in mitochondrially derived liposomes created by the membranes of mitochondria in (C) with additional lipids. Liposomes contain Phen Green to monitor the uptake of Cu. (F) Mitochondrial swelling rate in the presence of phosphate as a measure of phosphate uptake.

Fig 3: Metformin reduces EOC cell growth in an AMP-activated protein kinase (AMPK)-independent manner. (A) Epithelial ovarian cancer (EOC) cell lines that stably express sh-control or sh-TOM40 were harvested at 70% confluency. The expression levels of indicated proteins were measured by western blot analysis. (B) sh-TOM40 stably expressed EOC cells were introduced to si-Luciferase or si-AMPKa1 and then incubated in a CO2 incubator (5% CO2 and 37 ?) for 72 h. Cell viability was assessed by crystal violet assay (Left panel). Data in the bar graph are expressed as the mean of absorbance ± Standard Deviation (S.D.)., n = 16. # p > 0.05. The expression levels of indicated proteins including anti-AMPKa1 were measured by western blot analysis (Right panel). (C) Cell viability (relative absorbance at 550 nm) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. OVCAR-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 1 mM AICAR, 5 mM metformin, or 1 mM aspirin for 48 h. AICAR, 5-aminoimidazole-4-carboxamid ribonucleotide. (D) SK-OV-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 5 mM metformin for 48 h. Results are shown as the relative means ± S.D., n = 6; # p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (E) Western blot analysis of proteins expressed by EOC cell lines, OVCAR-3 and SK-OV-3, that stably express empty vectors or TOM40 and were cultured in dulbecco modified eagle medium (DMEM) with or without 25mM glucose and with or without 1 mM AICAR or 5 mM metformin for 24 h.

Fig 4: NMN supplementation and Parkin overexpression can reduce the accumulation of damaged neuronal mitochondria caused by PrP106-126.A, B Western blots of mitochondrial protein (TOMM40, COXIV, SOD2) expressions in N2a cells with A overexpressed and B knocked down (siRNA) parkin cells, with and without PrP106-126, nicotinamide mononucleotide (NMN), and urolithin A (UA) treatments (Tubulin and ß-actin served as loading controls.) C, D Comparisons of mitochondrial protein levels, relative to controls, in cells from A and B, respectively. Data were mean (SD). ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001. All experiments were repeated at least three times.

Fig 5: Expression of TOM complex in HT22 cells treated with Aß for 24 h. Cells were treated with 20µM Aß25 - 35 for 24 h, with or without followed by treating with 5µM DAPT or 20µM EUK 134 for 24 h. (A) TOM70, TOM40, TOM22, and TOM20 mRNA levels in HT22 cells after 24-h treatment with Aß25 - 35. (B) TOM70, TOM40, TOM22, and TOM20 protein levels in HT22 cells after 24-h treatment with Aß25 - 35. (C) Representative bands of western blot of TOM complex in Aß2535-treated HT22 cells. (D) TOM70 fluorescence of HT22 cells treated with Aß25 - 35 for 24 h. (E) TOM70 fluorescence relative intensity in HT22 cells after 24-h treatment with Aß25 - 35. (F) Representative bands of western blot of TOM70 in Aß25 - 35, DAPT, or EUK 134-treated HT22 cells. (G) TOM70 protein level in Aß25 - 35, DAPT, or EUK 134-treated HT22 cells. *P < 0.05, **P < 0.01, ***P < 0.001.