Af0639

The total protein of peritendinous tissues was isolated using cell lysis buffer (P0013, Beyotime Institute of Biotechnology, China) as per manufacturer’s instructions. Western blotting was performed following the standard protocol [17 (link)]. Antibodies against HO-1 (1:1000 dilution, ab68477, Abcam, UK), Nrf2 (1:1000 dilution, AF0639, Affinity Biosciences, USA), NF-κB (1:1000 dilution, Rabbit mAb #8242, CST, USA), p-NF-κB (Ser536) (1:1000 dilution, Rabbit mAb #3033, CST, USA), and β-Actin (1:2000 dilution, Rabbit mAb #4970, CST, USA) were used for Western blotting.

The proteins Nrf2 and PP65 were assessed in vitro. ATDC5 cells were seeded in 6-well plates and divided into the control, IL-1β, and IL-1β plus CDDO-Me (0.6 μM) groups. ATDC5 cells were pretreated with CDDO-Me for 24 h followed by stimulation with IL-1β (10 ng/mL) for 30 min. To assess Sox9 and MMP13 expression, ATDC5 cells were divided into the control, CDDO-Me (0.6 μM), IL-1β, and IL-1β plus CDDO-Me (0.6 μM) groups. The cells were treated with CDDO-Me (0.6 μM) or IL-1β or were treated with IL-1β plus CDDO-Me for 48 h. The cells were washed three times in PBS and then fixed with 4% paraformaldehyde for 30 min, followed by permeabilization using 0.1% Triton X-100 diluted in PBS for 20 min. Then, the sample was blocked with goat serum for 1 h at 37 °C and incubated overnight at 4 °C with the following primary antibodies: anti-MMP13 (1:1000, Cat.# AF5355, Affinity), anti-Sox9 (1:1000, ab185966, Abcam), anti-phospho-p65 (1:1000, Cat.# AF2006, Affinity), and anti-Nrf2 (1:1000, Cat.# AF0639, Affinity). Next, the samples were incubated with fluorescent secondary antibodies (1:100, Cat. BA1127, Boster) for 1 h at room temperature and labeled with DAPI (Cat. AR1176, Boster) for 30 min. Finally, the slide was sealed with 50% glycerin, and the fluorescence intensity was measured using ImageJ.

After micro-CT scanning, the samples were decalcified in 10% EDTA (pH = 7.4) for 4 weeks, then embedded in paraffin. Histological sections were prepared for hematoxylin and eosin (H&E) and TRAP staining. Immunofluorescence staining was performed with antibodies against TNF-α (1:100; AF7014; Affinity, Cincinnati, OH, USA), Nrf2 (1:100; AF0639; Affinity, Cincinnati, OH, USA), and HO-1 (1:100; AF5393; Affinity, Cincinnati, OH, USA). The images of stained slices were captured under a high-quality microscope (Leica DM4000B). The relative fluorescence of stained slices was quantified by ImageJ software (NIH, Bethesda, MD, USA). The number of OCs and TRAP-positive multinucleated OCs per field (Oc.S/BS) were calculated.