Enhanced chemiluminescence

Cells and muscle tissue were lysed in RIPA buffer (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein lysates were heated at 95°C for 5 min in 5 × SDS sample buffer, then separated by 10% SDS polyacrylamide gel electrophoresis (30 μg each lane), followed by transfer to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States) using a Mini Trans-Blot Cell system (Bio-Rad). The membrane was blocked with 5% non-fat milk for 3 h. The primary antibodies were incubated overnight at 4°C. The membranes were washed and incubated with secondary antibodies for 1 h at 37°C followed by visualization by enhanced chemiluminescence (Bio-Rad). Primary antibodies specific for EZH2 (ab3748, 1:1,000; Abcam, Cambridge, United Kingdom), MyoD (sc-760, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, United States), MyoG (sc-12732, 1:200; Santa Cruz Biotechnology), MyHC (sc-376157, 1:3,00; Santa Cruz Biotechnology), Ki67 (ab16667, 1:1,000; Abcam), p21 (sc-6246, 1:1,000; Santa Cruz Biotechnology), and β-actin (sc-4777, 1:1,000; Santa Cruz Biotechnology), along with goat anti-mouse IgG-HRP (sc-2005, 1:3,000; Santa Cruz Biotechnology) and goat anti-rabbit IgG-HRP (sc-2004, 1:3,000; Santa Cruz Biotechnology) secondary antibodies were used to detect protein expression.

Primary chondrocytes (5 × 10⁵/well) were seeded on a six-well culture plate. After incubation for two days, the cells were treated with different concentrations of CF and 10 ng/mL IL-1β at 37 °C for 24 h. The cells were digested in RIPA buffer containing the inhibitors for phosphatases (Millipore, Burlington, MA, USA) and proteases (Millipore, 535140). The total protein lysates (20 µg) were separated by 8% or 10% SDS-PAGE gel and transferred onto a PVDF for 90 min at 100 V.
After blocking with 5% nonfat skim milk for 1 h at room temperature, the membrane was incubated with specific primary antibodies (Table 1) at 4 °C overnight and then specific secondary antibodies for 2 h at room temperature. The proteins were detected using Enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA) and exposed to an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The protein expression level was quantified using ImageJ.

All samples were mixed with loading buffer and subjected to 10% SDS–polyacrylamide gel electrophoresis. Each lane was loaded with 50–100 μg of equal amounts of protein. All samples were then transferred onto PVDF membranes and blocked with 5% milk in Tris-buffered saline–Tween 20 (TBST) buffer for 1 h at room temperature. The membranes were subsequently exposed to first antibody against TFAM (29 kDa) and β-actin (42 kDa) (Abcam Inc, USA) at the dilution of 1:1,000 in 5% milk in TBST for overnight at 4 °C, and then membranes were washed and incubated with secondary antibody. Antibody binding was detected using enhanced chemiluminescence (BioRad). The results were quantified by densitometry using Image J software.

The cells were washed in 1× phosphate-buffered saline (PBS) and lysed in SDS lysis buffer containing 1 × PhosSTOP protease inhibitor (Roche) and a protease inhibitor cocktail (Roche). Equal amounts of the cell lysates were separated using SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Millipore). Nonspecific binding was blocked through incubation with 3% bovine serum albumin at room temperature for 1 h. Subsequently, blots were probed with primary antibodies at 4 °C, followed by incubation with appropriate secondary antibodies. Signals were visualized using enhanced chemiluminescence (Bio-Rad). The antibodies used in this study are listed in Supplementary Table 6.

Cardiomyocytes were collected and lysed after which equivalent quantity of protein was loaded and separated by 10% SDS-PAGE. Then all the separated protein was electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membrane was blocked by 5% skim milk for 1h, incubated with primary antibodies (Sirt4, 1:1000, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA; GAPDH, 1:2000, Cell Signaling Technology, Boston, USA) at 4°C overnight. Afterwards horseradish peroxidase-conjugated secondary antibody (1:10,000, Abcam, LA, USA) was also incubated on the membrane at 37°C for 1 h. Proteins were scanned and detected by enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) using a ChemiDoc MP system (Bio-Rad Laboratories). Image J software was used to analyze densitometric results of band.