Ru486

Human UC-MSCs were cultured in 12-well plates, upon reaching 80–90% confluency, cells were washed with PBS and then treated with indicated stimulations. The stimulations were TNFα (eBioscience, MA, USA) or IFNγ (eBioscience, USA) alone or a combination of these two cytokines (10 ng/ml each) for 24 h; TNFα (10 ng/ml) and IFNγ (10 ng/ml) combined with BAY 11-7082 (5 μM) (Selleck, WA, USA) or DMSO (Sigma, MA, USA) for 24 h; TNFα (10 ng/ml) and IFNγ (10 ng/ml) combined with RU486 (3 μM) (Selleck, USA) or DMSO for 24 h; Dexamethasone (10 ng/ml) (Sigma, USA) combined with RU486 (3 μM) or DMSO for 24 h.

To screen the Prestwick library mdx FAPs were seeded on 384 well plate at the density of 1,500 cells/well. 24 hours after seeding cells were treated for 6 additional days with the 1,120 compounds of the Prestwick library at the concentration of 5 μM. In vitro treatment of FAPs with 50 nM of TSA results in the inhibition of FAP adipogenic differentiation^{8 (link),10 (link)}. In our experimental settings, 20 nM of TSA were sufficient to inhibit mdx FAP adipogenic differentiation (preliminary experiments, data not shown) and therefore DMSO and 20 nM of TSA have been used as negative and positive controls respectively. Compounds were transferred from a 10 mM DMSO stock solution to assay plates by acoustic droplet ejection (ATS-100, EDC biosystems, USA). Cells were stained with ORO to visualize adipocytes while Hoechst 33342 was used to stain nuclei. Adipogenic differentiation has been assessed as the total pixel intensity (TPI) for ORO signal normalized for the number of nuclei in each field (ORO^norm). Adipogenic differentiation has been reported in Table S1 as: $$\left(OR{O}^{norm}\mathrm{Treated}/OR{O}^{norm}\mathit{DMSO}\right)⁎100$$
For validation studies, cells were treated with budesonide, dexamethasone and/or mifepristone (RU-486, Selleck Chemicals, #S2606) dissolved in DMSO, at various concentrations and administered at specific times, as indicated in the figure legends.