Fig 2: Inhibition of Drp1 enhanced the invasion of GH3 cells via activating STAT3. (A) The expression of STAT3 protein was detected by western blotting in four GH3 cell groups (VE, VE+Mdivi-1, Drp1++/++, Drp1++/+++Mdivi-1) after 48 hours (n = 3) (left panel). Statistical analysis of the western blotting results (right panel). (B) Nuclear pSTAT3Y705, a phosphorylated form of STAT3 and mainly involved in nuclear transcription, was detected by western blotting in the four groups. H3 was used as a reference (n = 3) (left panel). Statistical analysis of the western blotting results (right panel). (C) Mitochondrial p-STAT3S727, another phosphorylated form of STAT3 and mainly distribute in mitochondria, was detected by western blotting in the four groups. COX-IV was used as a reference (n = 3) (left panel). Statistical analysis of the western blotting results (right panel). (D) Expression of STAT3 protein levels in NIGHPAs (n =8) and IGHPAs (n = 8) samples were assessed by western blotting (left panel). Statistical analysis of the western blotting results (right panel). (E–G) IHC staining of STAT3, pSTAT3Y705, pSTAT3S727 in NIGHPAs samples (n = 10). (H–J) IHC staining of STAT3, pSTAT3Y705, pSTAT3S727 in IGHPAs samples (n = 10). (K) Statistical analysis of the IHC results. (L–Q) Activity of MMP2/9 were detected by situ zymography fluorescence staining in six groups (VE, VE+Mdivi-1, VE+Mdivi-1+HO-3867, VE+Mdivi-1+Cry, Drp1++/++, Drp1++/+++Mdivi-1). (R) Statistical analysis of the MMP2/9 fluorescent intensity. (S–U) GH3 cell invasion were evaluated by transwell assay when treated with Mdivi-1, Mdivi-1+HO-3867, Mdivi-1+Cry. (V) Statistical analysis of the invasive GH3 cell number in the three groups. HO-3867, a broad spectrum inhibitor of STAT3 phosphorylation. Cry, a selective inhibitor of STAT3 phosphorylation at Tyr705. (E–J, S-U), Scale bar =50 µm. L-Q, Scale bar =25 µm. *P < 0.05, **P < 0.01, ***P < 0.001. Data were expressed as mean ± SEM.

Fig 3: The protective effect of metformin is attributed to DRP1 phosphorylation but not AMPK activation in primary neurons. (A) Representative blots and densitometric quantification of AMPK protein in primary neurons. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to ß-ACTIN (bottom). (B) Representative blots and densitometric quantification of AMPK protein in primary neurons stimulated high glucose in the presence of GSK 621 or Compound C. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to ß-ACTIN (bottom). (C) Representative blots, and densitometric quantification of DRP1 protein in primary neurons stimulated high glucose in the presence of GSK 621, or Compound C. The phosphorylation of DRP1 (top blots) was normalized to total DRP1 protein (middle), and total DRP1 was normalized to ß-ACTIN (bottom). Fluorescent signals of Mitotracker-Red CM-H2X ROS (D) (200 ×, Bar = 800 µm) and quantification (E) in primary neurons stimulated with high glucose in the presence of GSK 621, or Compound C. Data presented as means ± SEM, *p < 0.05 vs cells under control condition, #p < 0.05 vs cells stimulated with high glucose by One-way ANOVA.

Fig 4: The proposed pathway through which extracellular succinate induced mitochondrial fission in a GPR91-dependent manner in cardiomyocytes.Under hypoxic and ischemic conditions, accumulated succinate was released to extracellular milieu. Extracellular succinate activated GPR91 receptor and its downstream PKCd/ERK1/2 signaling cascades. Succinate increased the translocation of Drp1 to mitochondria via PKCd activation, and induced MFF phosphorylation via ERK1/2 activation both in a GPR91-dependent manner, leading to mitochondrial fission

Fig 5: Succinate promoted Drp1 recruitment to mitochondria by GPR91-dependent PKCd activation.a–g Neonatal rat ventricular myocytes (NRVMs) were pretreated with or without rottlerin upon sodium succinate stimulation for 2 h. a Increased Drp1 phosphorylation (Ser 585) expression in whole cell detected by western blot. b Drp1 phosphorylation (Ser 585) in H9c2 cells transfected with PKCd siRNA. c Views of Drp1 recruitment to mitochondria with confocal microscope. Scale bar: 10 µm. d Mitochondrial Drp1 expression detected by western blot. e Recruitment of Drp1 to mitochondria in H9c2 cells transfected with PKCd siRNA. Bar: 10 µm. f PKCd/Drp1 interaction determined by co-immunoprecipitation and western blot. g Representative confocal images of mitochondrial morphology (upper, scale bar: 10 µm; lower, magnified images, bar: 5 µm). h Confocal images of PKCd translocation to mitochondria (upper, scale bar: 10 µm; lower, magnified images, bar: 5 µm) in H9c2 cells transfected with GPR91 specific or control siRNAs. i Western blot of Drp1 phosphorylation (Ser 585) expression in H9c2 cells transfected with GPR91 specific or control siRNAs. Data are shown as mean ± SD of five independent experiments; *p < 0.05 vs sodium succinate-only treatment; #p < 0.05 vs indicated treatment