Fig 2: EZTG improves the level of mitophagy in the ovaries of aged mice. (A,B) Immunofluorescence images of the PINK1 and Parkin. (C) Representative protein bands of PINK1 and Parkin. (D,E) Quantification of Immunofluorescence staining for PINK1 and Parkin. (F,G) The corresponding quantitative statistical results of PINK1 and Parkin protein. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, All data are expressed as the mean ± SD and at least three independent experiments were performed

Fig 3: Representative Western blots and OD bar chart analysis for the 24 h effect of treprostinil (blotted as “+”) on mitochondrial regulating proteins: (A) p62, (B) beclin1, (C) LC3 II, (D) mitofusin1, (E) mitofusin2, (F) parkin, and (G) PINK1. Bars represent means ± S.E.M. of at least three independent Western blots. The p-values were calculated by a paired Student’s t-test.

Fig 4: Silencing TFAM enhanced the sensitivity of HepG2 cells to sorafenib by inhibiting mitophagy. The changes in mitochondria were observed after sorafenib intervention for 24 h in HepG2 cells, with or without TFAM knockdown. (A) Upon TFAM knockdown, the mitochondrial morphology was detected by TEM in sorafenib-treated HepG2 cells under hypoxia conditions. The black arrows indicated normal mitochondria, and the red arrows indicated the damaged mitochondria. The left scale bar: 2.0 μm; the right scale bar: 500 nm. (B) Mitophagy-related proteins (parkin and pink1) were analyzed by Western blot. (C) Flow cytometry measured the effect of TFAM knockdown on MMP levels in hypoxia. HepG2 cells were treated with 5 μM CCCP for 24 h. The experiment was repeated three times. (D) Plate cloning assays showed that combination of 5 μM CCCP and TFAM knockdown under hypoxia conditions significantly promoted the inhibitory effect on cell proliferation. Data are expressed as mean ± SEM, where * p < 0.05, ** p < 0.01, and *** p < 0.001 denote statistical significance.

Fig 5: Remobilization increased the activation of mitophagy in rectus femoris within the first three days. (A) Mitochondrial biosynthesis was upregulated during remobilization to counteract this adverse effect. Mitochondria were generally normal in morphology and number in group C. A large amount vacuolated mitochondria and noted loss of mitochondria number were detected in group I-2w. Accumulation of mitophagosomes was observed in group R-1d and R-3d. (B) (a) The PGC-1α/GAPDH, HSP60/GAPDH, COX IV/GAPDH of the rectus femoris in each group (n = 3 per group). (b) The average protein level for. aP < 0.05 vs. group C, bP < 0.05 vs. group I-2w, cP < 0.05 vs. group R-1d, dP < 0.05 vs. group R-3d, eP < 0.05 vs. group R-7d. (C) (a) The PINK1/GAPDH, Parkin/GAPDH, Beclin-1/GAPDH, BNIP3/GAPDH, LC3B-II/LC3B-I of the rectus femoris in each group. (n = 3 per group). (b) The average protein level for. aP < 0.05 vs. group C, bP < 0.05 vs. group I-2w, cP < 0.05 vs. group R-1d, dP < 0.05 vs. group R-3d, eP < 0.05 vs. group R-7d. All the gels were trimmed and the samples derive from the same experiment and that gels/blots were processed in parallel