Bmpr2

The antibodies of CD9, CD63, CD81, and bone morphogenetic protein receptor 2 (BMPR2) were obtained from Abcam (Cambridge, MA, USA). Monocrotaline was purchased from the Fluorochem (Hadfield, Derbyshire, UK). The agomir and antagomir were obtained from Biomics Biotech (Nantong, China).

Total protein separated from hADSCs using a protein extraction kit (Bio-Rad, USA), and the concentration of total protein was determined using BCA kit (Tianjin, China). Subsequently, the protein (50 μg) was separated by SDS-PAGE. Then, moved to PVDF (Millipo,USA), the membrane was incubated with blocking buffer at room temperature for 1 h. Then, incubate the first antibody GAPDH (Abcam), Aggrecan (Proteintech), SOX9 (Proteintech), COL2A1 (Proteintech), BMPR2 (Abcam) overnight, followed by secondary antibody at room temperature for 1 h. The GAPDH was used internal control. All western blot experiments were repeated at least three times.

Western blot analysis was performed according to a previously described standard method (37 (link)). The primary antibodies used for the Western blot analyses were as follows: 14-3-3ζ (Santa Cruz Biotechnology, SC-293415, diluted at 1:500), E-cadherin (Abcam, ab133597, diluted at 1:5,000), N-cadherin (Abcam, ab76057, diluted at 1:1,000), vimentin (Abcam, ab137321, diluted at 1:1,500), BMP2 (Abcam, ab214821, diluted at 1:1,000), BMPR2 (Abcam, ab124463, diluted at 1:500), Smad1 (Cell Signaling Technology, #6944, diluted at 1:1,000), Smad5 (Cell Signaling Technology, #9517, diluted at 1:1,000), phosphorylated (p)-Smad1/5 (Cell Signaling Technology, #9516, diluted at 1:1,000), ID1 (Santa Cruz Biotechnology, SC-374287, diluted at 1:500) and β-actin (used as the loading control; Sigma, A1978). Western blot bands were quantified by ImageJ software (U.S. National Institutes of Health, USA). The experiments were repeated three times.

Cells were lysed (1% Triton-X100, NaF 100 mmol/L, NaPPi 10 mmol/L, Na₃VO₄ 1 mmol/L in PBS, supplemented with Complete anti-protease cocktail; Roche) for 20 min at 4 °C and sonicated. A total of 20 µg of protein was separated by SDS-PAGE (4–20% TGX-denaturing gels, BioRad) and transferred to PVDF membranes (Amersham, Sigma-Aldrich, St Quentin Fallavier, France). Blots were probed with TGFBR1 (Abcam, Cambridge, UK ab235178, 1/1000), cleaved PARP (#9541, 1/1000, Cell Signaling, Danvers, MA, USA), cleaved caspase-7 (#9491, 1/1000, Cell Signaling), BMPR2 (ab130206, 1/1000, Abcam), and GAPDH (MAB374, EMD Millipore, Burlington, MA, USA). Proteins were visualized with enhanced chemiluminescence using the LAS4000 microscopy imaging system, and densitometry analysis was performed using ImageJ Software (National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov, 1.53t).

Human tissue samples were fixed in 3.7% paraformaldehyde for 2 days and decalcified in a solution of 14% ethylenediaminetetraacetic acid (EDTA) in distilled water, pH 7.4 for 4 to 6 weeks at 4 °C. Samples were embedded in paraffin wax and serially sectioned (5 µm) (n = 8–15). OA cartilage damage was evaluated on Safranin-O Fast Green stained tissue slices by a Mankin’s score (range 0–14) [59 (link)].
Immunohistochemistry was performed with mouse monoclonal antibody to Grem-1 (Abcam, dilution 1:50), BMP-4 (Abcam, dilution 1:50), VEGFR-2 (Abcam, dilution 1:50), BMPR-1a (Abcam, dilution 1:50), BMPR-1b (Abcam, dilution 1:50), BMPR-2 (Abcam, dilution 1:50), as primary antibodies. Enzyme-induced antigen retrieval was performed as follows: 0.2 mg/mL hyaluronidase in PBS, pH 5.5, for 10 min at 37 °C and then 0.1 mg/mL pronase in PBS, pH 7.4, for 20 min at 37 °C. The R.T.U Vectastain kit (Vector) was used for detection, followed by counterstaining with Mayer’s hematoxylin. Irrelevant control antibodies (Dako) were incubated at the same concentration to assess non-specific staining. Preparations were mounted in Eukitt medium.