Fig 2: Activation of PGC-1a via ZLN005 inhibits cisplatin-induced mitochondria damage in vitro. (A) For the measurement of mitochondrial ROS (mtROS), HK2 cells were stained with Mito-SOX Red (2.5 µM) for 15 min at 37° C and determined by confocal microscope. Scar bar: 20 µm. (B) Mitochondrial ROS (mtROS) were measured by incubation with Mito-SOX Red. (C) The mitochondrial membrane potential measurement was detected with JC-1 (5 nM). Scar bar: 20 µm. (D) The expression of mitochondria-related proteins (ATP5b and Ndufs4) was measured by western blotting. (E) ATP content was measured by using an ATP Assay, ATP concentration was calculated in nmol/mg protein, and the data are represented as the rate of control. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01 vs. Con; &P < 0.05, &&P < 0.01, &&&P< 0.001 vs. Cisp. (Con, control; Zln, ZLN005; Cisp, cisplatin; C+Z, cisplatin + ZLN005).

Fig 3: Silencing of TFEB partially abolishes the protective effects of ZLN005 in cisplatin-treated HK2 cells. HK2 cells were transfected with control siRNA (sicon, black column) or TFEB siRNA (sitfeb, white column) for 6 h and treated with cisplatin in the presence or absence of ZLN005 for 48 h. (A) The expression of autophagy-related protein (TFEB, P62 and LC3) was measured by western blotting. (B) The expression of mitochondria-related proteins (ATP5b and Ndufs4) was measured by western blotting. (C) Mitochondrial ROS (mtROS) were measured by incubation with Mito-SOX Red. (D) The expression of apoptosis-related proteins was measured by western blotting. (E) The effects of ZLN005 on cisplatin-induced apoptosis were determined by flow cytometry. (F) ATP levels were measured by using an ATP Assay kit. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con; &P < 0.05, &&P < 0.01 vs. Cisp; #P < 0.05, ##P < 0.01 vs. sitfeb. (Con, control; Zln, ZLN005; Cisp, cisplatin; C+Z, cisplatin + ZLN005).

Fig 4: DNJ augments the level of OPA1 oligomers and improves its biomolecular function.(A) Schematic identifying the molecular target of DNJ combining pulldown and mass spectrum-proteomics analysis. (B) The Coomassie staining gel of eukaryotic purified Flag-His-OPA1 is shown. (C) Immunoblot confirmation of the DNJ-binding protein with purified Flag-His-OPA1. (D) Immunoblot confirmation of the endogenous DNJ-binding protein in cell lysis using OPA1 antibody. (E) MST assay for the affinity between DNJ and purified EGFP-OPA1 protein. (F) HCM cybrids were treated with DNJ for 8 hours and collected to purify the mitochondria. Mitochondria were incubated with 10mM EDC for 30 minutes. Proteins were separated by SDS-PAGE and immunoblotted using anti-OPA1 antibodies. (G) HCM cybrids treated with DNJ for different time periods (1, 2, 4, 6, and 8 hours). The above mitochondria were then treated as in (F). Proteins were separated by SDS-PAGE and immunoblotted using anti-OPA1 antibodies. (H) Mitochondria were isolated from cybrids and treated with DNJ or DMSO for 30 minutes. The above mitochondria were then treated as in (F). (I) Graphical illustration of OPA1 function in mitochondrial cristae remodeling. (J and K) Endogenous co-IP assay using IgG and OPA1 antibodies was performed to detect OPA1-IMMT (J) and OPA1-ATP5B (K) interactions.