Silencing of Transcription Regulators

ON-TARGETplus SMARTpool siRNAs targeting Arid5a, HuR, TRAF2 and MCPIP1 were from Dharmacon (Lafayette, CO). For RNA silencing, ST2 cells and MEFs were seeded overnight in antibiotic-free α-MEM, and N/TERT2G cells were seeded in KC-serum free media (Gibco) with supplements. Transfection was performed 18 h later with 50nM siRNAs with DharmaFECT Reagent 1. Culture media was replaced after 24 h, and IL-17 administered 24 h later. Flag/Myc- Arid5a was from OriGene (Rockville MD). All other constructs were described (6 (link), 22 (link), 27 (link), 63 (link)). Transfections of ST2 cells and MEFS were performed with Fugene HD (Promega, MadisonWI) or CaPO₄ (HEK293T cells). RNA was prepared with RNeasy Kits (Qiagen, Valencia CA). cDNA was synthesized by Superscript III First Strand Kits (ThermoFisher, Waltham MA). Quantitative real-time PCR (qPCR) was performed with the SYBR Green FastMix (Quanta Biosciences, Beverly MA) and analyzed on a 7300 ABI Real Time instrument. Primers were from QuantiTect Primer Assays (Qiagen).