Figure 2. SIRT3 siRNA knockdown in normal chondrocytes promotes SOD2 and OGG1 hyperacetylation, generation of mtDNA⁴⁹⁷⁷ deletion mutation, and mitochondrial dysfunction.

Cultured normal human chondrocytes (passage 1) was transfected with SIRT3 siRNA and control siRNA for 48 hours. Cell lysates were subjected to Western blot analysis for expression of SIRT3, acetylation status and expression of SOD2 and OGG1 (A, top panel), and subunits of mitochondrial respiratory complex III and V (B). PCR detected mtDNA⁴⁹⁷⁷ deletion mutation (A, bottom panel). Mitochondrial DNA content was determined as ratio of mtDNA (COXI or COXII) to nuclear DNA (18S rDNA) by PCR (C). Oxygen consumption (D) and intracellular ATP was quantified (E) as described in the Methods. Data in A and B represent 3 individual experiments with 3 different normal donors. Student t-test was used for statistical data analysis in C, D and E comparing control siRNA with SIRT3 siRNA chondrocytes (n=3 donors with 3–4 replicates for each donor, 2 males and 1 female, age 33, 44 and 39, respectively). The mean differences, 95% CIs and p values for COXI/18S dDNA, COXII/18S rDNA, oxygen consumption and ATP levels were 0.61 ± 0.04, 0.51 to 0.7, p<0.0001, 20.27 ± 1.52, 0.51 to 0.7, p<0.0001, and 0.46 ± 0.01, 0.43 to 0.5, p<0.0001, respectively.