Rho kinase inhibitor

mSGOs are structures derived from SGOs, containing differentiated cells of the SG. In order to make mSGOs, SGOs were first generated in self-renewal conditions, either in control SGPC medium or with additional 1 μM Iso. At the end of the passage, gels were dissociated using Dispase, collected by centrifugation, and re-suspended in 25 μL of SG medium. This suspension was then mixed with 50 μL of growth-factor-reduced Matrigel and deposited as a drop in the center of a 12-well plate. After incubation for 20 min at 37° to solidify, 1 mL of enhanced SG medium was added, with 1 μM Iso, or 1 μM Met, as required. Enhanced SG medium was composed of 20 ng/mL epidermal growth factor (EGF) (Sigma-Aldrich), 20 ng/mL fibroblast growth factor 2 (FGF2) (Sigma-Aldrich), N2 (Invitrogen), 10 mg/mL insulin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), and 10 μM Rho Kinase Inhibitor (Abcam). Cultures were maintained for 14 days, and medium was supplemented at day 3 and day 6 (0.5 mL of medium volume added). mSGO formation efficiency was calculated by quantifying the number of structures with at least three branches terminating in round end buds. mSGOs were enumerated as a proportion of seeded number of SGOs.

Cell Culture medium, Dulbecco’s modified Eagle medium (DMEM), and Keratinocyte serum-free medium (SFM) with supplements including EGF (Epidermal Growth Factor) and Bovine Pituitary Extract (BPE), penicillin-streptomycin, trypsin, Dulbecco’s Phosphate Buffered Saline (DPBS) and Sodium pyruvate were purchased from Gibco (ThermoFisher, Cramlington, UK). Y-27632, Rho kinase inhibitor, and Sulforhodamine B (SRB) assay kit were purchased from Abcam, UK. Dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) were purchased from Sigma, UK. The GenElute RNA/DNA/Protein Purification Plus kit was purchased from Sigma-Aldrich, Gillingham, UK.