P acc

Cells and freshly isolated mitochondria were washed with cold PBS and lysed in the RIPA buffer (Sigma–Aldrich, U.S.A.). Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, U.S.A.). Proteins (30 μg) were subjected to SDS/PAGE (10%) and then electrotransferred on to a nitrocellulose membrane. Transferred proteins were then incubated with primary antibodies against PGC1-α, NRF1, Tfam, LC3B I, LC3B II, p62, beclin-1, AMPK, p-AMPK, acetyl-CoA carboxylase (ACC), p-ACC, OPA1, NADH ubiquinone oxidoreductase (complex I), and succinate dehydrogenase (complex II) (Abcam, U.S.A.). β-actin, α-tubulin, and VDAC1 were loaded as the internal references. Bands were visualized using the goat anti-rabbit IgG-HRP secondary antibody (1:2000; Abcam, U.S.A.). Bands were developed using chemiluminescent substance (Thermo Scientific, U.S.A.).

The proteins suspension of liver tissues were obtained using a total protein extraction kit (APPLYGEN, Beijing, China) and protein concentrations were determined by the BCA protein assay kit while using BSA as a standard. Subsequently, standard western blot procedures were followed, as described elsewhere [41 (link)]. β-actin was used as an endogenous control and blots were quantified by using Image J Software (NIH, Bethesda, MD, USA). The primary and secondary antibodies used for western blot (AMPK, AMPK-p, p-ACC, ACC, CD68, and β-actin) were purchased from Abcam (Cambridge, UK) and other antibodies (CaMKKβ, NF-κβ, TGF-βR1, PPARα, SMAD2/3-P, and SMAD2/3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Oil red dye, hydrogen peroxide (H₂O₂), and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (St Louis, MO, USA). FFA free bovine serum albumin (BSA) was obtained from Merck (Germany). Detection kits for SOD and MDA were from NJJCBIO Inc. (Nanjing, China), while commercial detection kit for triacylglycerides (TG) was taken from Beijing BHKT (Beijing, China). ROS detection kit was used from Beyotime (Haimen, Jiangsu, China). The Takara quantitative RT-PCR kit and SYBR Green Premix Ex Taq were products of Takara Biomedicals Inc. (Shiga, Japan). The primary and secondary antibodies used for Western blot (AMPK, AMPK-p, p-ACC, and β-actin) were purchased from Abcam (Cambridge, UK). Compounds C and AICAR were obtained from Tocris Bioscience (Minneapolis, MN, USA). Cytokine array kit was purchased from RayBiotech, Inc. (Norcross, Ga.) All other laboratory chemicals were of the highest purity from commercial suppliers.

Total protein was extracted from WT and db/db cardiac tissues using RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk in TBS with 0.1% Tween 20, membranes were incubated with the following primary antibodies overnight at 4 °C: primary antibodies were purchased from Cell Signaling (TGF-β1, PGC1a, NRF1, NRF2, total OXPHOS, ACC, p-ACC Ser⁷⁹, ACSL1, ATGL, FOXO1, p-FOXO1 Ser²⁵⁶, AMPK, p-AMPK Thr¹⁷², Akt, p-Akt Ser⁴⁷³, mTOR, p-mTOR Ser²⁴⁴⁸, PPARγ1/2, NF-κB p65, β-Actin, and GAPDH); Abcam (GLUT4, Col1a1, and TFAM); Invitrogen (FABP3, IGFBP7, and p-SREBP1 Ser³³⁸); and Santa Cruz Biotechnology (CD36, SREBP1, MFN1, OPA1, and DRP1). After washing, the membranes were incubated with the HRP–conjugated secondary antibody (Jackson ImmunoResearch, USA), diluted in 5% skim milk, and incubated for 1 h at room temperature. Finally, the membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20. Immunodetection was performed using an enhanced luminol-based chemiluminescent substrate (WESTSAVE Up, AbFrontier, Korea). GAPDH and β-Actin were used as loading controls. Quantification of each band was performed using ImageJ software.

Cells were lysed radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) that was blocked with 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h, then incubated overnight at 4 °C with primary antibodies against α-SMA (1:200), SM22α (1:250), AMPK (1:200), p-AMPK (1:200), ACC (1:250), p-ACC (1:250), and GAPDH (1:1000) (all from Abcam). The membrane was then incubated with horseradish peroxidase-conjugated IgG for 1 h at room temperature, and protein bands were visualized with a chemiluminescence kit (Beyotime Institute of Biotechnology) and X-ray imaging system (Tanon, Shanghai, China). Signal intensity was quantified with ImageJ software.

To analyze the PAI-1, p16, phosphorylated AMP-activated protein kinase (AMPK) α and phosphorylated acetyl-CoA carboxylase (ACC) proteins, C28/I2 chondrocytes were lysed in ice-cold PBS with protease/phosphatase inhibitor. Cell lysates were collected, quantitated, and employed for western analyses. For this purpose, a total of −20 µg of the proteins were loaded onto a 4–20 percentage precast polyacrylamide gel electrophoresis (PAGE) gel (Bio-Rad, USA) and blotted with specific antibodies including PAI-1 (diluted 1:2000; Abcam, USA), P16 (diluted 1:1000; Abcam, USA), p-AMPK (diluted 1:1000; Abcam, USA), AMPK (diluted 1:3000; Abcam, USA), p-ACC (diluted 1:1000; Abcam, USA), and subsequently probed with secondary antibody (diluted 1:3000; Abcam, USA). Reactive bands were detected using ChemiDoc™ reagent (Bio-Rad Laboratories, Hercules, CA, USA).