A11418

BCA kit (P0012, Beyotime, China) was used to quantify the extracted protein. After quantification, it was then electrophoresed in 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, USA). After blocking with 5% skim milk, the primary antibody was incubated at 4 °C overnight, and the secondary antibody was incubated at room temperature for 1 h. ECL kit (Tanon, China, 185001) and chemiluminescence instrument (Tanon, China) were used for imaging. The primary antibodies against were listed as follows: MMP13 (18165-1-AP; 1:2000; Proteintech), MMP3 (A11418; 1:1000; Abclonal), Col2a1 (ab307674; 1:1000; Abcam), ADAMTS5 (bs-3573R; 1:1000; Bioss), GPX4 (67763-1-Ig; 1:1000; Proteintech), Nrf2 (ab307026; 1:5000; Abcam), P53 (ab26; 1:500; Abcam), SLC7A11 (ab175186; 1:5000; Abcam), NF-κB p65 (8242T; 1:1000; CST), p-NF-κB p65 (3035; 1:1000; CST), and GAPDH (A19056; 1:10000; Abclonal).

After removing the medium, NPCs were washed with PBS three times and lysed in lysis buffer (Beyotime, Jiangsu, China) with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Beyotime) and phosphatase inhibitor cocktail I (Sigma, USA). Cells were disrupted using an ultrasonic cell disruptor (Sonics & Materials, Inc., USA). The lysates were then centrifuged at 15000 × g for 15 min to collect the supernatants. The protein concentration was determined using a BCA protein assay kit (Boster, China). After being denatured using SDS‒PAGE denaturation buffer (Servicebio, China), the supernatants were loaded onto 10% or 15% SDS‒PAGE gels. Western blotting was performed as previously described.^[70
] The following antibodies were used: Collagen Type II (Col2, 1:1000, A1560, Abclonal, China), Aggrecan (Acan, 1:1000, A11691, Abclonal, China), Sox9 (1:1000, ab185966, Abcam, UK), MMP3 (1:1000, A11418, ABclonal, China), MMP13 (1:1000, A11755, ABclonal, China), Adamts5 (1:1000, A2836, Abclonal, China), Bcl2 (1:1000, 26593‐1‐AP, Proteintech, USA), Bax (1:1000, 50599‐2‐lg, Proteintech, USA), caspase3 (1:1000, 19677‐1‐AP, Proteintech, USA), LC3 (1:1000, 12741T, CST, USA) and GAPDH (1:3000, ab181602, Abcam, UK).

Western blot assay was performed as described by Sale et al [19 (link)]. Total proteins were extracted from cells using RIPA lysate (R0010, Solarbio) containing phenylmethylsulfonyl fluoride protease inhibitor (PMSF, P0100, Solarbio). Then, protein concentration was detected with a BCA kit (PC0020, Solarbio). After separated by 5-10% SDS-PAGE, transferred to the PVDF membrane, blocked by nonfat milk (A600669, Sangon Biotech, China), proteins on the membrane were incubated overnight at 4°C with the primary antibodies: iNOS (1: 1000, A0312, Abclonal, China), COX-2 (1: 2000, A1253, Abclonal), MMP-1 (1: 2000, A1191, Abclonal and 1: 1000, DF6325, Affinity), MMP-3 (1: 2000, A11418, Abclonal), MMP-13 (1: 2000, A11148, Abclonal and 1: 1000, AF5355, Affinity), p-ERK1/2 (1: 1000, AF1015, Affinity, China), ERK1/2 (1: 1000, AF0155, Affinity), p-p38 (1: 500, AF4001, Affinity), p38 (1: 500, AF6456, Affinity), p-JNK (1: 1000, AF3318, Affinity), JNK (1: 500, AF6318, Affinity), p-p65 (1: 1000, AF2006, Affinity), p65 (1: 1000, AF5006, Affinity), and GAPDH (1: 10,000, 60,004-1-Ig, Proteintech, China). Next, the membrane was incubated with appropriate secondary antibody. Finally, membranes were detected by Western electrogenerated chemiluminescence (ECL) Substrate (D1010, Solarbio) and the images were analyzed by Gel-pro analyzer software (Media Cybernetics, CA, USA).