Kc serum free media

ST2, HEK293T and mouse embryonic fibroblasts (MEFs) were cultured in α-MEM (Sigma Aldrich, St. Louis MO) with L-glutamine, antibiotics and 10% FBS (11 (link)). N/TERT2G cells were cultured in KC-serum free media (Gibco) supplemented with 30 μg/ml bovine pituitary extract- BPE, 0.2 ng/ml recombinant human epidermal growth Factor- EGF, CaCl₂ and antibiotics. Murine IL-17 was used at 200 ng/mL, human IL-17 at 100 ng/mL and murine TNFα at 5μg/mL (Peprotech, Rocky Hill, NJ). The IKK inhibitor VII (EMD Millipore, Burlington MA) was used at 10 μM, and Actinomycin D (Sigma, St Louis MO) at 5μg/mL.

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).