Fig 2: Key mitochondrial oxidative metabolism enzyme levels had no significant change but activities in LPS treated WT mice is significantly reduced. (A) Representative immunoblots of succinate dehydrogenase (SDH-A 70 kDa), citrate synthetase (CS 52 kDa), cytochrome c oxidase IV (COX IV 17 kDa), and loading control prohibitin (32 kDa). Densitometric quantification of (B) CS, (C) SDH-A, (D) COX IV, and (E) CS activity, (F) NADH:ubiquinone oxidoreductase activity, and (G) succinate dehydrogenase activity in hearts from WT and sEH null mice. Data presented as mean ± SEM, N = 5. Statistics: Two-way ANOVA with Tukey’s post hoc test. *p < 0.05 vs. control, #p < 0.05 vs. WT LPS treatment groups.

Fig 3: SMYD5 is not involved in mitochondrial fission and fusion or autophagy. (A) Western blots of proteins critical for mitochondrial functions (TFAM, SDHA, COX IV, UCP2, UCP3) in primary IECs isolated from SMYD5fl/fl and SMYD5?IEC mice. GAPDH was used as loading control. Left: each blot represents 1 mouse of specific genotype (n = 3). (B) RT-qPCR analysis of Sod1 and Sod2 mRNA levels in primary IECs isolated from SMYD5fl/fl and SMYD5?IEC mice. n = 3. (C) Western blots of protein expression of SOD2 in primary IECs isolated from SMYD5fl/fl and SMYD5?IEC mice. GAPDH was used as loading control. Each blot represents 1 mouse of specific genotype (n = 4 for SMYD5fl/fl mice; n = 5 for SMYD5?IEC mice). (D) Western blots of proteins critical for mitochondrial dynamics (mitofusin [MFN]1, MFN2, dynamin-related protein 1 [DRP1], LC3) in SMYD5 KO or parental HCT116 cells. SMYD5 protein abundance was examined to validate KO efficacy. GAPDH was used as loading control. (E) Western blots of FUN14 domain containing 1 (FUNDC1) expression in primary IECs isolated from SMYD5fl/fl and SMYD5?IEC mice. GAPDH was used as loading control. Each blot represents 1 mouse of specific genotype (n = 3 for each genotype).

Fig 4: B[a]P metabolism was not involved in IF1 up-regulation in response to B[a]P. F258 cells were pre-treated for 1 hour with a-NF (10 µM) prior to co-exposure to B[a]P for 48 hours. The involvement of B[a]P metabolism in IF1 up-regulation was assessed by analyzing IF1 protein level on mitochondrial fractions by western blotting. Histogram gives the results of densitometric analysis of IF1 protein level relative to ß-F1 subunit protein level. COXIV was used as loading control. Results were representative of 3 independent experiments. *p < 0.05, ***p < 0.001, DMSO vs B[a]P-treated cells, unless otherwise quoted.

Fig 5: Effect of high fat diet (HFD) versus low fat diet (LFD) on the relative abundance of mitochondrial Complex IV protein, COXIV, in single fibers of each fiber type. The loading control was myosin heavy chain (MHC). There were no significant differences between LFD and HFD for any fiber type. Values are means ± 95% confidence interval.