Osteogenic differentiation assay

BMMSCs were cultured in osteogenic medium containing 100 nM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate (all from Sigma-Aldrich). Different doses of IL-12, IL-17, IL-23, and INF-γ (all from Peprotech, Rocky Hill, NJ, USA) were added. To inhibit apoptosis, a pan-caspase inhibitor (Z-VAD-FMK, 20 μM) and a caspase 3 inhibitor (Z-DEVD-FMK, 20 μM, both from Selleckchem, Houston, TX, USA) were used to treat BMMSCs at 4 h before IFN-γ and IL-17 were added. SiRNA for Fas and TRAIL was transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. The target sequence of Fas mRNA was 5′-CCCGAGAAUUGCUGAAGACAU-3′, that of TRAIL mRNA was 5′-UCUCGGAAAGGGCAUUCAUUU-3′, and that of scramble siRNA (control) was 5′-UUCUCCGAACGUGUCACGU-3′. The medium was changed every 3 or 4 days. To assess osteogenic differentiation, these cultures were stained with Alizarin Red S (Sigma-Aldrich). Finally, the calcium precipitates were dissolved in 0.1 N sodium hydroxide and quantified using a Tecan Safire² microplate reader (Tecan, Durham, NC, USA) by absorbance at 548 nm.