Integrin αv

Monosialoganglioside GM1 and N-Hexanoyl-biotin-Monosialoganglioside GM1 (GM1-biotin) were purchased from Matreya LLC (State College, PA, USA). GM1-Pentasaccharide was obtained from Carbosynth (Compton, UK). An antagonist for chemokine receptor CCR2 (RS 102895) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). D-(+)-Mannose, N-Acetyl-D-galactosamine, D-(+)-Galactose and D-(+)-Glucose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Heparin was obtained from JW Pharmaceutical (Seoul, Korea). Antibodies were purchased as follows: Anti-Arginase (Arg) 1, mannose receptor (CD206), YM1, MCP-1, IL-10, interferon gamma (IFN-γ), IL-2 receptor gamma (γC), and integrin αV were purchased from Abcam (Cambridge, UK). Anti-p-STAT3, p-STAT5, TNF-α, and IL-1β were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p-STAT1, p-STAT6, STAT, STAT3, STAT5, STAT6, F4/80, VEGF, and GAPDH were purchased from Santa Cruz Biotechnology Inc. Anti-β-actin was obtained from Sigma-Aldrich. Anti- nitric oxide synthase (iNOS) was purchased from Millipore (CA, USA).

BM-MSCs were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor cocktail and insoluble debris was removed from the supernatant lysate by centrifuging at 15,000 × g for 15 min. Protein concentration was examined using BCA Protein Assay (Thermo Scientific, Waltham MA). 50 μg of total protein was loaded in each lane of 10% SDS-PAGE and transferred to nitrocellulose membranes. After the non-specific binding was blocked using 3% non-fat milk in TBST, membranes were incubated with primary antibodies against ALP (1:1000 dilutions, Abcam, UK), BMP4 (1:20000 dilutions, Abcam, UK), RUNX2 (1 µg/ml, Abcam, UK), Spp1 (1.25 µg/ml, Abcam, UK), Colla1 (1 µg/ml, Abcam, UK), FNDC5 (1:3000 dilutions, Abcam, UK), Irisin ((1:3000 dilutions, Abcam, UK), integrin αv (1:5000 dilutions, Abcam, UK) and GAPDH (1:1000 dilutions, Abcam, UK) overnight at 4 °C. Membranes were then washed three times with 0.1 M PBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Blots were developed and detected using Pierce ECL chemiluminescent substrates (Thermo Scientific, Waltham, MA).

Western blotting was performed according to a previously described protocol 10 (link). The cell pellet was washed with cold PBS once and then lysed with RIPA buffer (Thermo Scientific) containing protease inhibitor, and phosSTOP phosphatase inhibitor cocktail (Roche, Welwyn Garden, Swiss, UK). The lysates were centrifuged at 14,000 rotations per minute for 15 minutes at 4°C, and then the cleared supernatant was collected. The proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose membranes and probed with primary antibodies. The primary antibodies used for western blotting were against integrin β3 (Abcam, Cambridge, UK), integrin β1 (Abcam, Cambridge, UK), integrin αv (Abcam, Cambridge, UK), phosphor-mTOR (Abcam, Cambridge, UK), mTOR (Abcam, Cambridge, UK), phosphor-ERK1/2 (Cell Signaling Technology, USA), ERK1/2 (Cell Signaling Technology, USA), phosphor-Akt (Cell Signaling Technology, USA), Akt (Cell Signaling Technology, USA), Bcl-2 (Proteintech, China), Bax (Proteintech, China), cleaved caspase 3 (Cell Signaling Technology, USA) , PTEN (Cell Signaling Technology, USA) and β-actin (Sigma, USA). The immunoreactive bands were visualized using an ECL reagent (Pierce, Rockford, IL, USA).

Mifepristone and progesterone were obtained from the pharmacy department of Yueyang Hospital of Integrative Traditional Chinese and Western Medicine; hematoxylin-eosin, Antifade Polyvinylpyrrolidone Mounting Medium, and nitric oxide (NO) assay kit were purchased from Beyotime (China); the antibodies ERα, ERβ1, PR, Integrin αV, Integrin β3, OPN, LIF, eNOS, and p-eNOS were purchased from Abcam (UK); vascular endothelial growth factor (VEGF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Proteintech (USA); Alexa Fluor 488 nm, Alexa Fluor 555 nm, Akt, p-Akt, and β-tubulin were purchased from CST (USA); Lycopersion Esculentum Lectin was purchased from Vector Laboratories (USA); DAPI was purchased from Sigma (USA); chemiluminescence detection kit was purchased from Millipore (USA).