Fig. 4

STAT3 knockdown prevented uric acid-induced mitochondrial fission and dysfunction and oxidative stress and alleviated ATP production in AC16 cardiomyocytes, and HMGCS2 overexpression reversed these effects. AC16 cells were first treated with a combination of siRNA (si NC or si STAT3) and plasmids (OE NC or OE HMGCS2) under different conditions and then treated with uric acid (400 mg/L). A, B Representative western blot image (A) and the corresponding statistical analysis (n = 5/group) (B) of P-STAT3, T-STAT3, HMGCS2, FIS1, MFN1, MFN2 protein levels. C, D Mitochondrial morphology in AC16 cells treated with uric acid (400 mg/L, 24 h) was assessed using MitoTracker Red. Representative images (C, scale bar: 10 µm) and ImageJ quantification (D) of the number of mitochondria (count), the mean perimeter (perimeter), the mean aspect ratio (aspect ratio), the mean form factor (form factor), the number of mitochondria branches/mitochondria (Branches/mito) and the branches length/mitochondria (Branch length/mito) are shown. E, F Mitochondrial membrane potential was analyzed using JC-1 staining. Representative fluorescence images of JC-1 aggregates (red) and monomers (green) are presented (E, scale bar: 25 µm), along with quantification of the JC-1 aggregate/monomer ratio (F, n = 5/group). G, H (G) Representative images of DHE staining. and the corresponding statistical analysis (n = 5/group). Scale bar: 50 µm. I Relative MDA content in AC16 cells (n = 5/group). J Relative SOD activity in AC16 cells (n = 3/group). K. Relative ATP content in AC16 cells (n = 5/group). The data represent the means ± S.E.M.s. *p < 0.05 vs the indicated group