Cytokine and Drug Treatment of BMSCs

Before RNA isolation, BMSCs were incubated with or without cytokines of (50 ng ml^–1 IL-17, 10 ng ml^–1 IFNγ, and 10 ng ml^–1 TNFα) or drugs (100 nM RA and 10 μg ml^–1 BetA), respectively, or jointly for 6 h (Han et al., 2014 (link); Song et al., 2015 (link)). Total mRNA was isolated with MagZol^TM Reagent (R4801-03, Magen) according to the manufacturer’s instruction. mRNA purity and quantity were determined with NanoDrop (Thermo Scientific) before qPCR and RNA-seq analysis. For Real-Time qPCR, cDNA was synthesized from mRNA by using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit (AT341, Transgen). Quantitative Real-Time PCR was performed on Bio-Rad CFX96 Touch^TM Real-Time PCR Detection system with SYBR Green I Master Mix reagent (11203ES03, YEASEN). Sequences of forward and reverse primer pairs are as follows: