Lysis buffer

The cells were washed three times with PBS and suspended in ice-cold lysis buffer (Bio-Rad, Hercules, CA, USA). The lysates were separated by 12% SDS-PAGE, and the proteins of equal quantity were transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline with Tween-20 buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% nonfat milk (Yili, China) and incubated overnight at 4°C with collagen II, collagen X (Abcam, Cambridge, MA, USA), aggrecan, Bcl-2, Bax, caspase-3 (Santa Cruz, Dallas, TX, USA), p21, p27, CDK2, CDK4 (Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2, and ERK1/2 (Abcam, Cambridge, MA, USA) primary antibodies. The following day, the membranes were washed and incubated with the corresponding secondary antibodies at room temperature (22-28°C) for 1 h. An enhanced chemiluminescence detection system (Thermo Scientific, MA, USA) was finally used to determine the emission of the membrane. The experiments were performed in three independent replicates. The GAPDH expression level was used as an internal control. The Western blot results were quantified with an image analyzer (Quantity One-4,2,0, Bio-Rad, Hercules, CA) and were normalized to GAPDH immunostaining. The relative protein expression was compared with the control group.

SMMC7721 and HepG2 cells were stained with anti-MICA/B (clone 6D4, Biolegend, CA, USA) for 30 min and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG1κ mAb (BD Biosciences, NJ, USA) for 20 min on ice. Mouse IgG1κ isotype control (BD Biosciences, NJ, USA) was used as a negative control. A minimum of 20,000 gated events/sample was collected on a flow cytometer (FACSCalibur, Elite ESP, FL, USA) and analyzed using CellQuest software. SMMC7721 and HepG2 cells from different treatment groups were lysed in lysis buffer (Bio-Rad, CA, USA), and the western blot analysis was performed as described previously [10 (link)]. The cytotoxicity analysis was performed with the CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay (Promega, WI, USA), according to the protocol provided. To analyze the involvement of MICA/B in cytolytic activity of NK cells, anti-MICA/B mAb (6D4) or isotype-matched control Ab was added during the cytolytic assay.

Liver tissue was homogenized in lysis buffer (Bio-Rad) supplemented with protease inhibitors (Complete protease inhibitor cocktail, Roche) using a ratio of 1 mg tissue per 4 μl buffer. Samples were centrifuged at 500 g for 15 min and protein concentration determined in the supernatants. They were diluted with Bioplex sample diluent to a final concentration of 300 μg/ml. Plasma was obtained from whole blood taken from the left ventricle by cardiac puncture. Aliquots of 400–500 μl of blood were aspirated into a 2 ml syringe containing anticoagulant and immediately placed in EDTA containing tubes. Blood cells were separated by centrifugation at 1500 g for 15 min and the resulting plasma diluted 1:4 in Bioplex sample diluent. Cytokine content was analyzed using the Bio-Plex Multiplex immunoassay kit following the manufacturer's instructions.

For western blot analysis, the cells were plated in six-well plates and cultured as described above. After reaching confluency, the cells were treated with each agent. The cells were rinsed with ice-cold PBS and scraped on ice into lysis buffer (Bio-Rad, Hercules, CA, USA) containing 65.8 mM Tris-HCl (pH 6.8), 26.3% (w/v) glycerol, 2.1% sodium dodecyl sulphate (SDS), and 0.01% bromophenol blue, to which 2-mercaptoethanol was added to achieve a final concentration of 5%. The cell lysates were sonicated for 20 s. The cell lysates were electrophoresed on 10% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Bio-Rad). The blots were blocked with Tris-buffered saline (TBS) containing 1% Tween 20 (Bio-Rad) and 3% bovine serum albumin (BSA) for 1 h at 4 °C. Then, the blots were incubated overnight at 4 °C with gentle shaking with a primary antibody against phosphorylated ERK1/2 (1:2000 dilution) and other primary antibodies (1:1000 dilution). These blots were extensively washed with TBS containing 1% Tween 20 and were further incubated with a 1:5000 dilution of horseradish peroxidase-coupled IgG of specified animal species (rabbit or mouse) matched to the primary antibodies in TBS for 30 min at 4 °C. The blots were then washed, and the signal was visualised using an enhanced chemiluminescence technique.