Nb600 844

All rats were sacrificed after the pain behavior experiment. The knee joints from each of the rats were acquired and fixed with 5% buffered paraformaldehyde for 72 h, followed by decalcifying with 14% EDTA, lasting for a month. Each sample was then embedded in paraffin, sectioned (3 µm), and collected on microscopic slides. After staining with hematoxylin and eosin (HE) and Safranin-O (SO), the images of staining were pictured by a light microscope (Axio Scope A1, ZEISS, Germany) and analyzed with the Image-Pro Plus (IPP) 6.0 software (Media Cybernetics, MD, United States). Pathologic findings of the OA progression were evaluated by Mankin’s and OARSI scoring systems by double-blind observations (Mankin et al., 1971 (link); Glasson et al., 2010 (link)). Anti-Col2 (Novusbio, NB600-844, 1:500) antibodies were performed on a 3-μm-thick tissue section, and the immunoreactivity was semi-quantified by using the IPP software.

Expression of the hedgehog signaling molecules Patched (Ptc), Smoothened (Smo), and Gli1 was determined by western blotting. Expression of the cartilage-related proteins collagen II and ACAN was determined by western blotting following induction on days 10 and 21. Three samples from each group were randomly selected to extract cellular proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control protein. The primary antibodies for Ptc (1:500, orb19265) and Smo (1:1600, orb19363) were purchased from Biorbyt (Cambridge, UK). Gli1 (1:1000, ARP32368_T100) was purchased from Aviva Systems Biology (San Diego, CA, USA). Primary antibodies against Runx2 (1:500, ab23981) and collagen X (1:500, ab58632) were purchased from Abcam (Cambridge, UK). Primary antibodies against collagen II (1:200, NB600–844), ACAN (1:100, NB600–504), and GAPDH (1:2000, NB300–328) were purchased from Novus Biologicals (Littleton, CO, USA). HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, and HRP-conjugated anti-goat IgG antibodies were used as secondary antibodies at a 1:2000 dilution and were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Chondrocytes were lysed with immunoprecipitation lysis buffer (Sigma‐Aldrich, MO) to extract total protein. BCA Protein Assay Kit (P0010, Beyotime, China) was used for the quantification of total protein. Protein extracts were subjected to 10% SDS‐PAGE, and transferred to polyvinylidene fluoride membranes. After blockade of 3% BSA, the membranes were incubated with primary antibodies FoxO3 (ab109629, Abcam, UK, 1:1000), COL2A1 (NB‐600‐844, Novus, MO, 1:1000), Sox9 (ab185966, Abcam, UK, 1:5000), ACAN (A11691, ABclonal, China, 1:500), ADAMTS5 (ab41037, Abcam, UK, 1:1000), Runx2 (ab236639, Abcam, UK, 1:1000), Bcl‐2 (ab32124, Abcam, UK, 1:1000), Bax (ab32503, Abcam, UK, 1:1000), PCNA (ab92552, Abcam, UK, 1:1000), and GAPDH (NB100‐56875, Novus, MO, 1:5000) at 4°C for 12 hours, and incubated with HRP goat anti‐rabbit IgG (AS014, ABclonal, China, 1:2000). The membrane was detected using BeyoECL Star (P0018AS, Beyotime, China). GAPDH was used as the internal reference.

The proteins were obtained from NP cells by RIPA lysis and extraction buffer (Thermo Scientific). The protein concentrations were determined by using a BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's protocol. Western blot analyses were performed according to standard protocols. Briefly, the proteins were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gels. Then, transfer the gels to polyvinylidene fluoride (PVDF) membranes (Amersham, Buckinghamshire, UK). 5% skim milk was used to block the membranes for 2 h at room temperature, and incubate membranes overnight at 4°C with anti-STC1 antibody (AB_2608564, 1 : 1000, Invitrogen, USA), anti-VEGF antibody (AB_2212682, 1 : 1000, Invitrogen, USA), anti-MMP3 antibody (NB100-91878, 1 : 1000, Novus, USA), anti-Col II antibody (NB600-844, 1 : 1000, Novus, USA), anti-IL-1β antibody (AB_468396, 1 : 1000, Invitrogen, USA), and anti-β-actin antibody (abs118937, 1 : 3000, Absin, China) in 5% bovine serum albumin (BSA) in TBS-T overnight at 4°C. After washing with TBS-T three times, the membranes were incubated with the secondary antibody for 2 h at room temperature. Protein bands were detected by using the ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Inc., USA) according to the manufacturer's specifications.

Three‐dimensional chondrocyte pellets were transferred to microtubes, washed with PBS (−) and stored at −80°C. After adding 100 μL RIPA buffer (Nacalai Tesque, Kyoto, Japan) with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque), pellets were homogenized on ice until complete dissolution. Homogenized samples were centrifuged, and supernatants were collected. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) with an EnVision 2104 Multi plate reader (PerkinElmer, Waltham, MA, USA). The expression of each protein was detected using a 12–230 kDa or 60–440 kDa Separation Module with Wes (ProteinSimple, Minneapolis, MN, USA) automated capillary‐based immunoassay system called “Simple Western.” For each lane, 2 μg of protein at a concentration of 0.5 μg/μL, primary antibodies, and Anti‐Rabbit or Anti‐Mouse Detection Module (ProteinSimple) were loaded and ran according to the manufacturer's instructions. Antibodies were diluted with Antibody diluent II (ProteinSimple) to 1:100 for β‐actin (CST, #4970) and COL X (Abcam, Cambridge, UK; ab182563) and 1:50 for COL I (Novus, Littleton, CO, USA; NBP1‐30054) and COL II (Novus, NB600‐844).