Hif 2α

Cells were washed three times using cold PBS and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) containing protease inhibitors. Approximate 30 μg of protein was separated with 10% SDS–PAGE gel and blotted onto nitrocellulose membranes. Then membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with primary antibodies against GAPDH (Shanghai Kangchen), RICTOR (Bethyl Laboratories), VEGFA (Abcam), Akt (Cell signaling Technology), mTOR (Cell signaling Technology), HIF1α (Abcam) and HIF2α (Abcam) at 4°C overnight, followed by TBST wash and 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).

Cell lysates containing total protein (20 μg) were electrophoresed on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were blocked in 5% skim milk blocking buffer for 1 h and incubated with primary antibodies overnight at 4°C. Membranes were rinsed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The protein bands were visualized using enhanced chemiluminescence (ECL; Thermo Fisher Scientific). Primary antibodies used were the following: HIF-1α, HIF-2α, FAK-p³⁹⁷, and FAK (Abcam; Cambridge, MA, USA); PLOD2 (Proteintech; Wuhan, China), and β-actin (Beyotime, China).

C2C12 cells were harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Whole-cell protein extracts were quantified using the BCA assay, separated by SDS-PAGE 8–12%, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included Myogenin (Abcam, 1:1000, ab124800, Cambridge, MA, USA), MyoD (Santa Cruz, 1:200, sc-377460), HDAC9 (Abcam, 1:1000, ab59718), HIF1α (Abcam, 1:1000, ab179483), HIF2α (Abcam, 1:1000, ab179825), H3K9 (Abcam, 1:1000, ab32129), H3K14, H3K18, H4K16 (Cell Signaling, 1:1000), LC3I/II (Cell Signal, 1:1000, 12741), Beclin1 (Cell Signal, 1:1000, 3738), Atg5 (Cell Signal, 1:1000, 12994), Atg7 (Cell Signal, 1:1000, 8558), Atg12 (Cell Signal, 1:1000, 4180), p62 (Cell Signaling, 1:1000, 23214), p-GSK3β Ser9 (Cell Signal, 1:1000, 9323), GSK3β (Cell Signal, 1:1000, 12456), and active-β-catenin (Millipore, 1:800, 05–665). Stripped membranes were reprobed with GAPDH (Abcam, 1:4000, ab181602) as a loading control. Signal detection was performed using the ECL Kit (Beyotime Institute of Biotechnology) after incubation with an anti-rabbit or anti-mouse IgG secondary antibody (CoWin Bioscience Co., Beijing, China). Experiments were performed in triplicate.

Cells were seeded on gelatin-coated coverslips. After treatments, cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 15 min, washed with PBS, and permeabilized with 0.2% saponin (Sigma-Aldrich) and 1% fatty acid-free BSA (Sigma-Aldrich) in PBS, all at room temperature. After washing cells with PBS, they were stained overnight at 4 °C with Ki67 (sc-23900, Santa Cruz Biotechnology), HIF-1α (ab2185, Abcam, Cambridge, UK), HIF-2α (ab199, Abcam), or BNIP3 (sc-56167, Santa Cruz Biotechnology) antibodies. Then, cells were washed with PBS and incubated for 1 h at room temperature with Alexa 488-conjugated antimouse (Z25002, Molecular Probes, Eugene, OR, USA) or Alexa 647-conjugated antirabbit (Z25308, Molecular Probes) IgGs. Coverslips were washed with PBS and mounted on glass slides with fluorescent mounting medium Fluoroshield™ containing 4′6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) to be visualized in a Zeiss LSM 800 confocal laser scanning microscope (Zeiss AG, Jena, Germany). Confocal images were analyzed with ZEN software (Zeiss AG, Jena, Germany). Fluorescence quantification was performed using ImageJ software (NIH, Bethesda, MD, USA) and the corrected total cell fluorescence (CTCF) formula.

Western blot analysis was conducted as previously described (He et al., 2015). The primary antibodies were Nanog (1 : 1000, Cell Signaling Technology, Beverly, MA, USA, #4548), OCT4 (1 : 1000, Cell Signaling Technology, #2750), CD133 (1 : 1000, Cell Signaling Technology, #5741), HIF‐1α (1 : 1000, Abcam, Cambridge Science Park, Cambridge, USA, ab39266), HIF‐2α (1 : 1000, Abcam, ab72130), β‐actin (1 : 500, Abcam, ab92611), and BCRP (1 : 800, Abcam, ab26056). The bands were visualized by enhanced chemiluminescence. The band intensities were quantitatively analyzed using imagej Software (National Institutes of Health, Bethesda, MD, USA).

MicroCT (GE Locus SP) was used to access the bone mass, density, geometry, and trabecular microarchitecture of the right femurs from 6-week-old control and condition knockout (CKO) mice. Parameters computed from these data include trabecular thickness, number, separation, and connectivity at the distal femoral metaphysis and cortical thickness and cross-sectional area at the mid-diaphysis. The left femurs were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, paraffin embedded, and stained with H&E using standard methods. For immunohistochemistry, antigen retrieval was performed by boiling in 10 mM sodium citrate (pH 6.0) for 5 minutes. Sections were incubated with antibodies against HIF-1α (Abcam), HIF-2α (Abcam), VEGF (Novus Biologicals), PCNA (R&D Systems), and HO-1 (Abcam). The slides were examined using a Zeiss Axio microscope. Image-Pro Plus software was used to quantify the integrated optical density.

Wogonoside (purity >98 %), was purchase from Nantong Feiyu Biological Technology Co, ltd (Nantong, China). Carboxymethylcellulose (CMC) and type II collagenases were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant human IL-1β was purchased from PeproTech (NJ, USA). The primary antibody against collagen II, aggrecan, MMP-9, MMP-13, ADAMTS5, collagen X, HIF-2α, TRAF6, p-ERK1/2, ERK1/2, p-Stat3, Stat3 and GADPH were acquired from Abcam (Cambridge, UK), MMP-3 and iNOS antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). Sox-9 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Anti-VEGF-A, Anti-Runx-2, Anti-IRAK1, goat anti-rabbit, and anti-mouse IgG-HRP was from Bioworld (OH, USA) and antibodies against COX-2, PI3K(p110), PI3K(p85), AKT, p-AKT, p-IKKα/β, IκBα, p-IκBα, p65, and p-p65 were purchased from Cell Signaling Technology (Danvers, MA, USA); Alexa Fluor®488 labeled and Alexa Fluor®594 labeled Goat Anti-Rabbit IgG (H+L) second antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The 4', 6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Shanghai, China). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA).