Fig 2: 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 3: PGC-1a positively regulate the TFEB in HK2 cells. (A) HK2 Cell lysates were immunoprecipitated (IP) with an anti-PGC-1a or an anti-TFEB antibody, then immunoblotted (IB) with TFEB and PGC-1a antibodies. Anti- IgG antibody as a negative control. (B, C) The expression of TFEB and PGC-1a was measured by western blotting. (D) Luciferase activity in HK-2 cells transfected with the TFEB promoter-reporter construct linked to luciferase along with PGC-1a plasmids or control empty vectors. (E) Representative immunofluorescence images of TFEB in HK2 cells. Scar bar: 20 µm. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. overcon; #P < 0.05, ##P < 0.01, ###P < 0.001 vs sicon (Con, control; sicon, sicontrol; overcon, overcontrol).

Fig 4: Knockdown of CRIM1-inhibited autophagy under P4, E2 and IFN-t treatment. (A) EECs were infected with a lentivirus specific for CRIM1 and a negative lentivirus (shN) for 48 h. Western blot analysis of CRIM1 levels in EECs. (B) After being treated with P4, E2 and IFN-t, total proteins from EECs were subjected to Western blotting. (C,D) Fluorescence microscopy images of TFEB expression in EECs and histograms of fluorescence intensity. Representative images of three independent experiments are shown. Scale bar = 100 µm. (E) EECs were treated with P4, E2 and IFN-t for 12 h. The EECs were fixed and processed for TEM analysis. Scale bar = 2 µm. The data are presented as the means ± SEM of three independent experiments. * Significant difference (p < 0.05) compared with other groups; ** Significant difference (p < 0.01) compared with other groups.

Fig 5: ZLN005 treatment alleviates renal injury in cisplatin-induced AKI mice via PGC-1a/TFEB pathway. The male C57BL/6 mice were injected once with cisplatin (16mg/kg, i.p.) to induce AKI, followed by ZLN005 treatment (15mg/kg/d, i.g.) for 4 days. (A) Western blots of TFEB, P62 and LC3 levels in kidney. (B) Representative TEM micrographs of mouse renal tubular epithelial cell mitochondria from each group. Scale bar, 2 µm (wireframe indicates the magnified image). (C) The expression of mitochondria-related proteins (ATP5b and Ndufs4) was measured by western blotting. (D) For the measurement of mitochondrial ROS (mtROS), frozen sections of freshly renal tissues were stained with Mito-SOX Red (2.5 µM) 15 min at 37° C and determined by confocal microscope. Scar bar: 20 µm. 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 vs. Cisp. (NC, normal control; Cisp, cisplatin; C+Z, cisplatin + ZLN005).