Fig 2: IP3R inhibitor Xest-C suppressed mitochondrial Ca2+ and ROS accumulation in DNs with MFF insufficiency. Stem cells from human exfoliated deciduous teeth were differentiated into DNs in the absence or presence of Xest-C. (a) DNs were stained with Rhod-2 AM. The fluorescence intensity of Rhod-2 AM was measured using a plate reader. The mean ± SEM was taken from three independent experiments. n.s., not significant, * p < 0.05. (b) DNs stained with Mag-Fluo-4 AM were observed by confocal microscopy. Scale bar = 25 µm. The fluorescence intensity of Mag-Fluo-4 AM was measured using a plate reader. The mean ± SEM was taken from three independent experiments. n.s., not significant, * p < 0.05, ** p < 0.01. (c) IP was performed using an anti-MFF (upper panel) and anti-VDAC1 (lower panel) antibodies in untreated DNs. Immunoprecipitants were detected by Western blotting (WB) using the indicated antibodies. (d) Mitochondrial ROS levels were measured using flow cytometry. The mean ± SEM was taken from three independent experiments. n.s., not significant. ** p < 0.01.

Fig 3: Schematic model showing the mechanism of CSE/H2S regulated Drp1 activity in heart failure. During heart failure, CSE/H2S pathway was inhibited, whereas iNOS/NO pathways was potentiated. Although Drp1 S-sulfhydration and S-nitrosylation were competitively modified at the same cysteine, the effect of these two modifications on Drp1 activity was reverse. Aberrant activation of Drp1 translocated to mitochondria outer membrane and interacted with VDAC1 to cause mitochondria hyperfission and dysfunction.

Fig 4: a2A-AR/ß-arrestin2/NF-?B signaling contributes to mitochondrial dysfunction and cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative Western blotting of VDAC1 and COX IV in each group with densitometry analysis (n=6). (C) Representative Western blotting of VDAC1 and COX IV in TKPTS cells treated with PBS or NE (10 nM) for 48 hours with densitometry analysis (n=3–6 in each group). (D–F) TKPTS cells were transfected with scrambled siRNA (si-control) or ß-arrestin2 siRNA (si-Arrb2) for 24 hours and then incubated with NE (10 nM) for 48 hours. Mitochondrial ultrastructure was observed by transmission electron microscope (D). Scale bar, 1 µm. Mitochondrial mass was tested by MitoTracker deep red probe (100nM) staining (E). Scale bar, 50 µm. Mitochondrial membrane potential was examined by TMRE fluorescent dye (200 nM) (F). Scale bar, 20 µm. (G) Representative Western blotting of NF-?B p65 in TKPTS cells incubated with PBS, NE, and NE+BRL (10 µM) with densitometry analysis (n=4–6 in each group). (H) Representative Western blotting of NF-?B p65 in TKPTS cells of PBS, NE, and NE+si-control, NE+si-Arrb2 with densitometry analysis (n=3-4 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Bars represented means ± SEM.

Fig 5: BIRC6-236aa destabilizes the interaction between VDAC1 and CypD.A–C, BIRC6-236aa attenuates the interaction between VDAC1 and CypD. The interaction between VDAC1 and CypD or ANT1 was identified in a co-IP assay. BIRC6-236aa does not affect the interaction between VDAC1 and ANT1. Flag-BIRC6-236aa is overexpressed in IPEC-J2 cells. D and E, circBIRC6-2 weakens the interaction between VDAC1 and CypD. The interaction between VDAC1 and CypD was confirmed in a co-IP assay after overexpression and knockdown of circBIRC6-2, respectively. **p < 0.01, compared with the control (n = 3). CypD, Cyclophilin D; IP, immunoprecipitation.