Profiling RA Fibroblast-Like Synoviocyte Response

6 Primary RA FLS cell lines (highest ranked by RARα PPR: RA6, RA1, RA4 (CL1) and lowest ranked by RARα PPR: RA10, RA11, RA9 (CL2)) were cultured (at 5% CO2, 37 °C) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine, gentamicin, penicillin/streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) as previously described^{41 (link)}. Optimal RARα knockdown by siRNA transfection was performed as previously described^{42 (link)} and assessed in time course experiments. Cells were transfected with ON-TARGETplus SMARTpool siRNA targeting human RARα (Dharmacon) or ON-TARGET plus Nontargeting Pool siRNA (Dharmacon) using the Human Dermal Fibroblast Nucleofector Kit (Lonza) and cultured in 10%FCS for 3 days. These pools include multiple targeting or matched control siRNA sequences. Cells were serum starved for 18 h in 0.1%FCS/DMEM and subsequently treated with IL-1 (2 ng/ml) for 6 h. RNA was extracted using the RNeasy kit (Qiagen) and qPCR was performed using a cell-based standard approach⁴³ . Ct (threshold cycle) values were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH).