Fig 2: Tight junction disruption by EspF and Map is independent of mitochondrial function. (A) Constitutive expression of EGFP-EspF and EGFP-Map does not cause the release of cytochrome c from the mitochondria. Cytosolic and mitochondrial fractions were isolated from untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cell lines and analyzed by immuno-blotting with cytochrome c antibody. Equal loading on gels was confirmed by using COX IV as the mitochondrial marker and GAPDH as the cytosolic marker. Blots are representative of three independent experiments. Full-length blots are presented in Supplementary Figure S8. (B) Cells expressing EGFP-EspF and EGFP-Map display normal mitochondrial morphology. Cells from untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cell lines were grown on glass cover-slips and transduced with CellLight Mitochondria-RFP, BacMam 2.0 reagent to label the mitochondria with RFP. Cells were imaged after 16 hours on a Zeiss ApoTome (Axiovert 40 CFL) using 63 × oil objective. (n = 3); scale: 10 µm. (C,D) Cytotoxicity of EspF and Map to MDCK cells. Cytotoxicity was assessed by measuring the amount of LDH released by EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cells into the culture medium. Fold changes in LDH release with respect to EGFP cells are depicted (C). Results are representative of three independent experiments. Confluent cultures of untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cells were grown in the absence (0 µM) or presence (50 µM) of cisplatin to induce apoptosis (D). The number of viable cells was determined by MTT assay and fold change with respect to untreated cells was plotted for all cell lines. UT: Untransfected.

Fig 3: Bnip3 deficiency suppresses renal IR-induced mitophagy.Bnip3-KO and WT mice were subjected to renal IR or sham operation (sham). Renal cortex was collected for immunoblot analyses of LC3B-I/II, SQSTM1, TIMM23, and cyclophilin B (loading control), immunofluorescence of LC3B and COXIV, and for transmission electronic microscopy (TEM) analysis. a Representative blots. b–d Densitometry of LC3B-II (b), SQSTM1 (c), and TIMM23 (d). For densitometry, the target proteins signals were normalized to the cyclophilin B signal of the same samples to determine the ratios, and the ratios of sham-operated WT were arbitrarily set as 1 or 100%. e Representative images of immunofluorescence staining of LC3B (green) and COXIV (red) and DAPI (blue). f Percentage of renal tubules with LC3B puncta. g Percentage of renal tubules with mitophagosome formation (LC3B puncta colocalizes with mitochondria) in renal tubules. Bar: 50 µm. h TEM images of mitophagosome (pointed by arrowhead in the right panel) in renal proximal tubule cells after IR. Each symbol (circle and diamond) represents an individual mouse. Error bars: SEM, n = 3. *P < 0.05; **p < 0.01; ***p < 0.001

Fig 4: Analysis of PFF-induced TH+ neuron loss correlation with cortical mitochondrial protein levels. A Comparison of the number of SNc TH+ neurons determined by stereology to the level of the mitochondrial protein VDAC1 of the ipsilateral (left) and contralateral (right) cortex at 3 months p.i. Simple linear regression analysis revealed a significant correlation of TH+ neuron number and cortical VDAC1 levels in the contralateral hemisphere of PFF-injected mice. B, C No correlation was observed with COX-IV or DRP1 cortical protein levels and SNc TH+ cell number in either ipsilateral or contralateral hemisphere. D At 6-month p.i., while no significant correlation was observed between cortical COX-IV and SNc TH+ cell number, there was an apparent weak correlation in the contralateral hemisphere (right)

Fig 5: Lycopene counteracted mitochondrial dysfunction caused by H2O2. (A) Measurement of mitochondrial permeability transition pore opening. (B) Analysis of mitochondrial membrane potentials by flow cytometry. (C) Western blot analysis of Bax and Bcl-2 protein levels within mitochondria. (D) Quantification of Bax and Bcl-2 protein levels. *P<0.01 vs. control group. #P<0.01 vs. H2O2 group. NRFU, normalized relative fluorescence units; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-like protein 4; COX IV, cytochrome c oxidase subunit IV; H2O2, hydrogen peroxide.