Fig. 6

The effects of SS-31 on mitochondrial function and homeostasis were partially counteracted by inhibiting Sirt3/Pgc-1α. C57BL/6 mice received PBS + sham + DMSO, PBS + IS + DMSO, SS-31 + IS + DMSO, SS-31 + IS + 3-TYP or SS-31 + IS + SR-18292 treatment for 7 consecutive days, followed by sacrifice to evaluate mitochondrial function and mitochondrial homeostasis in TNC. A ROS levels were examined in different groups and normalized by total protein concentrations. n = 6 per group; One-way ANOVA. F(4, 25) = 7.078, ^***p = 0.0006; ^*p_{(PBS+IS+DMSO)} = 0.0222, ^**p_{(SS-31+IS+SR-18292)} = 0.0029, ⁺p_{(SS-31+IS+DMSO)} = 0.0259, ^##p_{(SS-31+IS+SR-18292)} = 0.0034. B ATP levels were examined in different groups and normalized by total protein concentrations. n = 9 per group; One-way ANOVA. F(4, 40) = 4.753, ^**p = 0.0031; ^**p_{(PBS+IS+DMSO)} = 0.0096, ^*p_{(SS-31+IS+3-TYP)} = 0.0301, ⁺p_{(SS-31+IS+DMSO)} = 0.0393. C Western blot analysis showed that expression of Sirt3 (F(4, 40) = 13.71, ^****p < 0.0001; ^****p_{(PBS+IS+DMSO)} < 0.0001, ^****p_{(SS-31+IS+3-TYP)} < 0.0001, ^****p_{(SS-31+IS+SR-18292)} < 0.0001, ⁺p_{(SS-31+IS+DMSO)} = 0.0153, ^##p_{(SS-31+IS+3-TYP)} = 0.0487, ^##p_{(SS-31+IS+SR-18292)} = 0.0216) and Pgc-1α (F(4, 40) = 15.15, ^****p < 0.0001; ^***p_{(PBS+IS+DMSO)} = 0.0002, ^****p_{(SS-31+IS+3-TYP)} < 0.0001, ^****p_{(SS-31+IS+SR-18292)} < 0.0001, ^##p_{(SS-31+IS+3-TYP)} = 0.0044, ^##p_{(SS-31+IS+SR-18292)} = 0.0039) in different groups. n = 9 per group; One-way ANOVA. (D) Representative immunoblots and quantification illustrated the levels of Tfam (F(4, 40) = 9.868, ^****p < 0.0001; ^***p_{(PBS+IS+DMSO)} = 0.0004, ^***p_{(SS-31+IS+3-TYP)} = 0.0001, ^***p_{(SS-31+IS+SR-18292)} = 0.0003, ⁺p_{(SS-31+IS+DMSO)} = 0.0417, ^#p_{(SS-31+IS+3-TYP)} = 0.0177, ^#p_{(SS-31+IS+SR-18292)} = 0.0363), Drp1 (F(4, 40) = 5.655, ^**p = 0.0011; ^***p_{(PBS+IS+DMSO)} = 0.0004, ^*p_{(SS-31+IS+SR-18292)} = 0.0492, ⁺p_{(SS-31+IS+DMSO)} = 0.0429), Fis1 and Pink1 in different groups. n = 9 per group; One-way ANOVA. E Immunofluorescence staining of VDAC1 and nucleus (DAPI) in TNC. The mean mitochondria length (F(4, 25) = 5.225, ^**p = 0.0034; ^*p_{(PBS+IS+DMSO)} = 0.0232, ^*p_{(SS-31+IS+3-TYP)} = 0.0410, ⁺p_{(SS-31+IS+DMSO)} = 0.0312) and area (F(4, 25) = 6.964, ^***p = 0.0007; ^*p_{(PBS+IS+DMSO)} = 0.0396, ⁺⁺p_{(SS-31+IS+DMSO)} = 0.0033, ^#p_{(SS-31+IS+3-TYP)} = 0.0150, ^##p_{(SS-31+IS+SR-18292)} = 0.0047) were calculated. Scale bar, 5 μm. n = 6 per group; One-way ANOVA. Data are represented as Mean ± SD; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 and ^****p < 0.0001 as compared to PBS + Sham + DMSO group. ⁺p < 0.05 and ⁺⁺p < 0.01 as compared to PBS + IS + DMSO group. ^#p < 0.05 and ^##p < 0.01 as compared to SS-31 + IS + DMSO group