Col2a1

The total proteins of ASC lysates were collected RIPA buffer (Signaling Technology, Inc.). The protein concentration was determined using a BCA kit (Beyotime, Shanghai, China). Total protein (30 µg/sample) was separated via 10% SDS-PAGE and to nitrocellulose membranes. Use 5% skimmed milk powder to block the membrans. The corresponding protein antibodies were as follows: COL2A1 (Abclonal, China, No. A1560; 1/500), ACAN (Abcam, USA, No. ab3778; 1/1000), Sox9 (Abcam, USA, No. ab285966; 1/1000), and β-actin (Boster, Chian, No. BM0627; 1/1000). Then, the membrane washing was performed with Tris-buffered saline/0.1% Tween (TBST) and incubated for 1.5 h with a HRP Goat anti-Rabbit IgG (Abcam, No. ab6721; 1/1000). The band visualization was carried out using the ECL system (Thermo Scientific, Rockford, IL, USA). β-actin used as internal control.

Recombinant murine IL-1β and recombinant murine M-CSF protein were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant murine IFN-γ was purchased from Peprotech (Rocky Hill, NJ, USA). Lipopolysaccharides (LPS) were obtained from Biosharp (Beijing, China). Metformin hydrochloride was purchased from CTAO (Eugene, OSU, USA). Anti-p85-PI3K, MTOR, p-MTOR, FoxO1, p-FoxO1, AKT, p-AKT, GSK3ß, p-GSK3ß, NF-κB, p-NF-κB, IκBα, p-IκBα, IκB kinase (IKK)-β, and p-IKKα/β antibodies were supplied by Cell Signaling Technology (Beverly, MA, USA). MMP3, MMP13, INOS, and CD206 antibodies were obtained from Abcam (Cambridge, UK). Col2a1 and Sox9 antibodies were purchased from Abclonal (Wuhan, China). IL-1ß, IL-6, CD86, and F4/80 antibodies were purchased from Proteintech (Wuhan, China). GAPDH, ß-Actin, and secondary antibodies were purchased from Boster (Wuhan, China).

The cells were lysed by RIPA (Solarbio, Beijing, China) with 1 mM PMSF (Solarbio, Beijing, China). Protein-extract supernatants were quantified using a BCA protein assay kit. A total of 20 μg protein was subjected to electrophoresis using SDS-PAGE and transferred to PVDF (Bio-Rad, Hercules, CA, USA). After blocking in 5% non-fat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against Col2a1 (ABclonal, Wuhan, China, 1:1000), Mmp13 (Proteintech Group, Rosemont, IL, USA, 1:1000), p16 (Abcam, Cambridge, UK, 1:1000), p21 (ABclonal, Wuhan, China, 1:1000), p62 (ABclonal, Wuhan, China, 1:1000), Lc3 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), Klf10 (Santa Cruz Biotechnology, Paso Robles, CA, USA, 1:1000), Bnip3 (Santa Cruz Biotechnology, Paso Robles, CA, USA, 1:1000), and β-actin (Proteintech Group, Rosemont, IL, USA, 1:5000), followed by incubation of the blots in HRP-conjugated secondary antibodies. The representative images were acquired by ECL reagents (Solarbio, Beijing, China) using an iBright 1500 imaging system (Thermo Fisher, Waltham, MA, USA).