A11755

The human and rat CEP tissues were cut into 4-μm-thick sections for Safranin O-fast green, hematoxylin and eosin (H&E) staining and IHC staining as previously described [17 (link)]. Sections were stained with H&E for cell density and morphology and with safranin O fast green for proteoglycans and matrix degeneration. The immunoreactivities of MIF, MMP13, and Col II were analyzed using an SP Rabbit & Mouse HRP Κit (CW2069, CWBIO, China). Rabbit anti-MIF (ab34712, Abcam), MMP-13 (A11755, ABclonal), and Col II (ab34712, Abcam) polyclonal immunoglobulin G antibodies were used at a dilution of 1:200. The sections were imaged using a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan). Three pathologists, who were blinded to the group, were responsible for counting the numbers of CEP cells under high-power fields (magnification × 200) for three sections in each specimen (N = 3 samples/group). The integrated optical density (IOD) was analyzed by Image Pro Plus 6.0.

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).