Fig 2: PEX protein colocalization with peroxisomes and mitochondria in baseline and mitophagy conditions, and roles of different PEX proteins in general autophagy A, BRepresentative images of TOMM20 and PMP70 co‐staining in HeLa/Parkin cells treated with the indicated siRNA 4 h after treatment with DMSO (A) or 10 μM CCCP (B). Scale bars, 20 μm.CRepresentative images of mitochondrial inner membrane protein ATP5B and peroxisome membrane protein PMP70 co‐staining in primary MEFs of the indicated genotype 24 h after treatment with DMSO or 30 μM CCCP. Scale bars, 20 μm.DQuantification of endogenous LC3 puncta in HeLa/GFP‐LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (˜100 cells analyzed per sample). Similar results were observed in three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.001, NS = not significant; one‐way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.

Fig 3: UA alleviated CuONPs-induced vascular injury in mice. A Schematic figure for animal experiments. C57BL/6 J mice were treated with vehicle (PBS) or UA (30 mg/kg) for 1 week by intragastric gavage (i.g.), and then exposed to CuONPs via intratracheal instillation (i.t.), meanwhile the mice in UA group and CuONPs + UA group were continuously treated with UA (30 mg/kg) by gavage for 3 days. B Representative TEM images of mice abdominal aorta dissected from CuONPs-treated mice. Mice were intratracheally instilled with CuONPs (5 mg/kg) for 3 days. N, nucleus; C, cytoplasm; Mt, mitochondria; IEM, internal elastic membrane. C and D Western blotting analysis and quantification of protein levels of Fis1, TIM23, ATP5B and LC3B in mice aorta tissues. Mice were intratracheally instilled with 2.5 mg/kg or 5 mg/kg CuONPs for 3 days. Actin was used as the internal control. E Representative TEM images of mice abdominal aorta dissected from CuONPs-treated mice. Mice were treated with UA (30 mg/kg) for 10 days by intragastric gavage and then intratracheally instilled with CuONPs (5 mg/kg) at day 7. N, nucleus; C, cytoplasm; Mt, mitochondria; IEM, internal elastic membrane. F Western blotting analysis and quantification of protein levels of mitochondria related proteins in aorta tissues dissected from CuONPs-treated mice with or without UA. Actin was used as the internal control. G Western blotting analysis and quantification of protein levels of oxidative stress response proteins in aorta tissues dissected from CuONPs-treated mice with or without UA. Actin was used as the internal control. In C and D, two-tailed unpaired Student’s t test was performed for statistical analysis. In F and G, one-way ANOVA with Tukey’s test was used for multiple comparisons. Results are representative of at least three independent experiments. Data are mean ± S.D. *p < 0.05

Fig 4: Mitophagy facilitates removal of damaged mitochondria in CuONPs-treated cells. A Representative immunofluorescence images of colocalization of GFP-LC3 and ATP5B in EA.hy926 cells treated with CuONPs (10 μg/ml). White arrows indicate colocalized dots of mitochondrial marker ATP5B with autophagosome marker GFP-LC3. Scale bars, 20 μm. B Western blotting analysis and quantification of protein level of LC3B, VDAC1, TIM23 and ATP5B in EA.hy926 treated with CuONPs (0, 5, 10, 15 and 20 μg/ml, respectively) for 12 h. C Western blotting analysis and quantification of protein level of LC3B, VDAC1, TIM23 and ATP5B in EA.hy926 treated with 10 μg/ml CuONPs for 0, 3, 6, 9 or 12 h. In (B) and (C), two-tailed unpaired Student’s t test was performed for statistical analysis. GAPDH was used as internal control. D The protein expression levels of VDAC1, TIM23 and ATP5B in EA.hy926 treated with or without wortmannin (Wort). Actin was used as internal control. E Representative FCM results of EA.hy926 cells labeled with MitoTracker Green FM. The cells were pretreated with or without chloroquine (CQ) for 1 h and then treated 10 μg/ml CuONPs for 12 h. MFI, mean fluorescence intensity. In D and E, one-way ANOVA with Tukey’s test was used for statistical analysis. All data are representative of at least three independent experiments. Data are mean ± S.D. *p < 0.05

Fig 5: The cell cycle-related protein AURKA shows physical proximity with core Complex V subunits, and its abundance is lowered after Complex V inhibition.A Representative western blot and corresponding quantification of the abundance of the cell cycle-related proteins AURKA and CCND1 in total lysates of MCF7 cells treated with oligomycin or Antimycin A for 48 h. Loading control: ACTB. Data are from n = 4 independent experiments, and presented as means ± SD. A.U.: arbitrary units. B, C FRET/FLIM analyses on MCF7 cells expressing B AURKA-GFP alone or together with ATP5F1A-mCherry or ATP5F1B-mCherry, or C AURKA-GFP alone or together with MCU-mCherry. Scale bar: 10 µm. Pseudocolor scale: pixel-by-pixel ΔLifetime. Graphs: ΔLifetime quantifications in the dotted areas. Data extend from minimum to maximum. n = 7 cells issued from one representative experiment (of three). *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001 compared to each corresponding control condition. ns: not significant.