Fak inhibitor 14

Rho was specifically inhibited using cell permeable C3 transferase (C3, Cytoskeleton, Denver, CO) and FAK was inhibited using FAK inhibitor 14 (SantaCruz Biotechnology, Dallas, Texas). Wnt/β-catenin was inhibited using cell-permeable FH535 ^{63 (link), 64 (link)} (SantaCruz Biotechnology, Dallas, Texas). hMSCs were seeded in a 8-well Labtek at a cell density of 8,000 cells per well and allowed to attach overnight (12 hours) in basal media, before media in the wells was replaced with serum-free media containing 0.5 μg/ml C3 transferase (C3) for 4 hours, or 1 μM FAK inhibitor 14 for 1 hour, or 15 μM FH353 for 1 hour. After pre-treatment, media was changed with induction media containing the inhibitor. Adipogenic media in AD conditions was cycled with 3 days induction followed by 1-day maintenance. Basal and osteogenic media was cycled every three days. Cell proliferation was analyzed 3 days post induction (time of descriptor analysis) using Alamar Blue assay using manufacturer’s protocol.

Hepatocytes were isolated from wild-type, 6–12 week-old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). In some experiments, hepatocytes were isolated from Hnf4a^fl/fl:Alb Cre⁺ mice that lacked HNF4α expression in adult hepatocytes and Hnf4a^fl/fl:Alb Cre⁻ wild-type littermates (kind gift from Dr. Frank J. Gonzalez) (17 (link)). All mice were cared for in accordance to the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.” Hepatocyte isolation was performed by two-step perfusion using Liver Perfusion and Liver Digest Media (Life Technologies, Pleasanton, CA) followed by separation with 50% Percoll (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient. Purity of live hepatocytes was routinely ≥90% by trypan blue exclusion. Hepatocytes were cultured in 5% fetal calf serum (Hyclone, Logan, UT) in DMEM supplemented with L-glutamine, antibiotics, insulin-transferrin-selenium, and HEPES (Mediatech, Manassas, VA). Inhibitors used and their final concentrations in cell culture were: 50μM Y-27632, 25μM FAK inhibitor-14, 0.5μM blebbistatin, or 20μM U-0126 (Santa Cruz Biotech, Dallas, TX). Unless otherwise noted, functional assays and gene expression analysis were performed 24h after hepatocyte plating.