Ly294002

hESCs were differentiated into neuroectoderm, endoderm, and mesoderm as described previously (Vallier et al. 2009 (link)). Briefly, cells were cultured in CDM supplemented with 10 µM SB-431542 (Tocris) and 12 ng/mL FGF2 for neuroectoderm; in CDM + PVA supplemented with 100 ng/mL Activin A, 20 ng/mL FGF2, 10 ng/mL BMP4, 10 µM Ly294002 (Promega), and 3 µM CHIR99021 (Selleck) for mesoderm; and in CDM − PVA supplemented with 100 ng/mL Activin A, 20 ng/mL FGF2, 10 ng/mL BMP4, and 10 µM Ly294002 (Promega) for endoderm. hESCs were differentiated as described before (Pauklin and Vallier 2013 (link)).

We used commercially available human pCRP (Lee BioSolutions, St Louis, MO, synthesized in E.Coli). pCRP was stored in 10 mM Tris-HCl (pH 7.5) with 2 mM CaCl₂ to prevent spontaneous formation of mCRP from pCRP. mCRP was prepared by treating pCRP with 8 M urea/10 mM EDTA for 1 h at 37°C as described [14] (link), [15] (link). We did not detect endotoxin in the pCRP used in this study using endotoxin detection kit (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific) (data not shown). mAb 7E3 (anti-human integrin β3) and mAb AIIB2 (anti-human integrin β1) hybridomas were obtained from ATCC. mAb SG73 (anti-human α4) hybridoma was a kind gift from K. Miyake (University of Tokyo). Anti-phospho-AKT (Thr-308), anti-phospho-ERK1/2, anti-ERK1/2, anti-AKT were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Cyclic RGDfV [16] (link) was purchased from Enzo Life Sciences (Plymouth Meeting, PA). BIO1211 was obtained from Tocris Bioscience (Ellisville, MO). LY294002 and PD98059 were purchased from Promega (Madison, WI). Chinese hamster ovary (CHO) cells that express WT β1, β3, or the β1–3-1 mutant have been described [17] (link). CHO cells that express human α4 have been described [18] (link). Recombinant soluble αvβ3 has been described [19] (link).

Minodronate was supplied from Astellas Pharmaceutical (Tokyo, Japan). Alendronate was purchased from LKT laboratories Inc. (St. Paul, MN, USA). These reagents were dissolved in phosphate-buffered saline (PBS; 0.05 M, pH7.4), filtrated through Syringe Filters (0.45 μm; IWAKI GLASS, Tokyo, Japan), and used for the various assays described below.
Farnesol (FOH) and geranylgeraniol (GGOH) were purchased from Sigma (St Louis, MO, USA). FOH and GGOH were dissolved in dry ethanol. U0126 and LY294002 were purchased from Promega (Southampton, Hants, UK) and dissolved in DMSO. The dissolved reagents were resuspended in PBS (0.05 M, pH 7.4) and filtered through Syringe Filters before use.