Fig 1: The knockdown of kindlin-2 eliminates the protective effect of irisin. The HL-7702 cells were transfected with siRNA of kindlin-2 (or negative control) for 48 h and treated with palmitic acid (PA, 0.2 mmol/L) and oleic acid (OA, 0.1 mmol/L) to induce lipotoxicity injury of hepatocytes. Cells were deprived of oxygen (94% N2, 5% CO2, 1% O2) in a serum-free and deoxyglucose-rich (5 mmol/L) medium to simulate hepatic ischaemia and hypoxia of mice for 1 h; then, the cells were changed to 5% CO2 condition with RPMI-1640 medium containing 10% FBS for 6 h. Western blot analysis of Bax and Bcl-2 (A) and their quantitative analysis of Bax (B) and Bcl-2 (C). DHE staining (red) (D) and its quantitative analysis of DHE fluorescence intensity (E). Western blot analysis of mitochondrial related proteins (F) and their quantitative analysis of Mfn-2 (G), ND3 (H), Tfam (I), ATPB (J), Drp-1 (K) and Fis-1 (L). Western blot analysis of ER stress-related proteins (M) and their quantitative analysis of GRP78 (N), CHOP (O), PDI (P) and Ero1-La (Q). Results are expressed as mean ± SE (n = 3-4/group) and compared by t test. NS P > .05
Fig 2: BBR and ROT promoted liver mitochondrial fusion. (A) Morphology and structure of mitochondria in liver with TEM. (B-D) The mitochondrial density (B), length (C) and area (D) by the statistical analyze of TEM (n =10). (E) Hepatic mtDNA copies (n = 7). (F) Citrate synthase activity of liver mitochondria (n = 6). (G) The protein levels of electron transport chain genes (CS, GRIM19, Ndufs4, mt-ND2, mt-ND3 and COXIV) by Western blot and relative signal strength quantification (n =3). Data were expressed as means ± SEM. *P < 0.05.
Fig 3: Incubation with rotenone reduces progressive motility and ATP levels. (A–D) Changes in sperm motility parameters evaluated using CASA: (A) sperm motility tracks, (B) motility of sperm of dairy goats. (C,D) Progressive motility and straight line velocity of sperm of dairy goats. (E–H) Effect of rotenone on sperm mitochondrial functions at the 5 h point: (E,F) opening fluorescence intensity of mitochondrial mPTP, (G) ATP content, and (H) immunolocalizations of COX-1 and COXVB in mitochondria. (I–M) Quantitative expression of COX-1, COXVB, ND3, and NRF1 using Western blotting after rotenone incubation for 5 h. All results are expressed as the mean ± standard error of the mean, with asterisks indicating statistical significance for the respective control group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, no significant difference.
Fig 4: The oxidative phosphorylation uncoupler FCCP reduces sperm motility and ATP content. (A–D) Motility parameters of dairy goat sperm using CASA: (A) motility tracks were generated using CASA. (B) Motility of sperm of dairy goats. (C,D) Progressive motility and straight line velocity of sperm of dairy goats. (E–G) Effect of FCCP on sperm mitochondrial functions: (E,F) opening fluorescence intensity of mPTP in mitochondria and (G) ATP content. (H–L) Expressions of COX-1, COXVB, ND3, and NRF1 determined using Western blotting after treatment with FCCP for 5 h. All results are expressed as the mean ± standard error of the mean, with asterisks indicating statistical significance for the respective control group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, no significant difference.
Fig 5: Analysis of complex I subunits encoded by mitochondrial and nuclear genes. A Western blot analysis of mtDNA encoding proteins. Twenty µg of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with ND6, ND4L, ND1, ND3 and ND5 antibodies, with ß-actin as a loading control. B Quantification of ND6, ND4L, ND1, ND3 and ND5 in C, CV0, CVND6, M, MV0, and MVND6 cell lines. The calculations were based on three independent determinations in each cell line. The error bars indicate standard error of the mean (SEM). P indicates significance based on Student’s t-test of the differences between M and MVND6 cell lines. C Western blot analysis of nucleus-encoding complex I subunits. Twenty µg of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with NDUFA10, NDUFS5, NDUFC2, NDUFA11, NDUFS2, NDUFS1 and NDUFB8 antibodies, with ß-actin as a loading control
Supplier Page from Abcam for Anti-MT-ND3 antibody