Fig. 6: Knockdown of Aifm1 or Chchd4 significantly reversed the protective effect of Sirt5 overexpression on NP cells under compression.

Rat NP cells were cultured in a compression culture chamber and subjected to 1 MPa static compression for 0 h or 24 h. Lenti-Sirt5 and siRNA of Aifm1 or Chchd4 were used to verify the protective effect of SIRT5 in rat NP cells under compression. Mitochondrial proteins were extracted from rat NP cells in each group. a–f Western blotting analysis and quantification of ATP5F1A, UQCRC2, SDHB, NDUFB8, and COX IV protein expression extracted from mitochondria (normalized to TOM20 expression, n = 3). g Quantification of ATP levels in NP cells in each group (n = 5). TOM20 IF staining was performed to visualize mitochondrial morphology. h Representative immunohistochemical staining of the mitochondrial structure in NP cells (n = 3). White scale bar = 10 μm; green scale bar = 2 μm. i Representative dot plot of cell apoptosis by flow cytometry analysis after Annexin V/PI dual staining (n = 3). j Quantification of the percentage of TUNEL-positive cells (n = 5). k–p Western blotting analysis and quantification of Aggrecan, MMP13, SIRT5, AIFM1, and CHCHD4 protein expression (normalized to β-actin expression, n = 3). Differences among multiple groups were analyzed by one-way ANOVA. The data in the figures represent the mean ± S.D. The P value is shown, *P < 0.05, **P < 0.01.