A19038

Samples were washed with PBS three times and then blocked with 5% BSA solution for 30 min, followed by incubation with the primary antibodies diluted in 5% BSA solution overnight at 4 °C: eNOS (ab5589, Abcam), alpha smooth muscle actin antibody (αSMA, ab7817, Abcam), CD68 (MCA341GA, BioRad), Cyclin D1 (A19038, ABclonal), Aggrecan (13880-1-AP, Proteintech), and CD206 (ab64693, Abcam). After that, samples were washed with PBS three times and incubated with goat anti-rabbit secondary antibody or goat anti-mouse antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Tissue sections without primary antibody incubation were used as negative controls. The stained samples were observed with an inverted microscope (IX73, Olympus, Japan). Five different tissue sections (n = 5) from five different rats for each group were quantified. Specifically, for CD68⁺ and CD206⁺ cell quantification, two different tissue sections from the same sample were stained with CD68 and CD206 antibodies, respectively. The numbers of the CD68⁺ and CD206⁺ cells and the ratio of CD206⁺ cells to CD68⁺ were then quantified. Five sets of tissues sections (n = 5) from five different rats for each group were quantified.

Total protein lysates were extracted with cell lysis buffer (P0013, Beyotime) containing a protease inhibitor (ST506, Beyotime) and quantified with a BCA assay kit (P0011, Beyotime). Protein samples were loaded on SDS‒PAGE gels and transferred onto polyvinylidene difluoride membranes. After being blocked in 5% BSA for 1 h at room temperature, the membranes were hybridized with primary antibodies against JMJD1C (1:1000; A20153, ABclonal, Shanghai, China), HK2 (1:1000; A0994, ABclonal), PGK1 (1:1000; A12686, ABclonal), LDHA (1:1000; A0861, ABclonal), p-STAT3 (1:1000; AF3293, Affinity), STAT3 (1:1000; AF6294, Affinity), cyclin D1 (1:1000; A19038, ABclonal), CDK4 (1:1000; A11136, ABclonal) or β-actin (1:1000; sc-47778, Santa Cruz, Dallas, TX, USA) overnight at 4 °C, followed by goat anti-rabbit IgG (1:5000; A0208, Beyotime) or goat anti-mouse IgG (1:5000; A0216, Beyotime) secondary antibody incubation for 45 min at 37 °C. The protein signals were visualized by an enhanced chemiluminescence system (P0018, Beyotime) and quantified using Gel-Pro-Analyzer Software (WD-9413B, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).