IL-17 and Synoviocyte Survival Figure 6. Synoviolin expression during chronic streptococcal cell wall-induced arthritis in wild-type (WT) mice. A representative image of synoviolin-positive staining diffusely (A) or in follicle-like structures of synovium (B and C, magnification X 200). Double immunostaining showing colocalisation of synoviolin (brown) and CD20 (blue) staining (D, magnification X 600) or IL-17 (blue) staining (E, magnification X 600) or isotype control (F, magnification X 600) in follicle-like structures of arthritic knee joints from WT mice. Synoviolin staining in synoviocytes infiltrating cartilage at the pannus-cartilage junction (G, magnification X 400). doi:10.1371/journal.pone.0013416.g006 as control cells for the RA FLS data. FLS or dermal fibroblasts were isolated by enzyme digestion and cultured in DMEM/10% FCS as described [3]. Cells beyond the third passage were .99% CD45 negative, and used between passages 4 and 9. Cultured RA FLS were treated with IL-17A or IL-17F 50 ng/ml (R&D Systems, Minneapolis, MN), IL-1b 0.1 ng/ml (Sigma-Aldrich), LPS 100 ng/ml (Sigma-Aldrich), TNFa 1 ng/ml (Biosource, Carmarillo, CA). IL-17RA or IL-17RC were antagonized with anti-IL-17RA mAb or anti-IL-17RC polyclonal goat Ab (R&D Systems). ERK, p38 and JNK MAPK were antagonized with PD98059 (50mM, chemicon)[42], SB203580 (5mM, chemi- con)[43], or SP600125 (50mM, chemicon)[44]. RNA interference Gene-specific small interfering RNA (siRNA) corresponding to a 19-nucleotide sequence targeting 1623–1641 of human IL-17RA (IL-17RA siRNA, NM_014339), 985–1003 of human IL-17RC (IL-17RC siRNA, NM_153460) or human synoviolin (Synoviolin siRNA, NM_032431) were purchased from Dharmacon (Lafay- ette, CO, USA). A non-targeting siRNA (siCONTROL) was used as a control for non-sequence-specific effects. RA FLS (16105) were transfected with 0.5 mg IL-17RA, IL-17RC, synoviolin siRNA or siCONTROL siRNA duplexes using a human dermal fibroblast nucleofector kit, protocol U-23 (Amaxa GmbH, Cologne, Germany)[23]. Apoptosis RA FLS were treated with sodium nitroprusside (sigma) 0.01– 0.5 mM for 24 h at 37uC. Apoptosis was analysed by annexin V expression (BD biosciences) by flow cytometry (Fluorescence activator cell sorter scan, Becton-Dickinson, Mountain View, CA) or fluorescence microscopy using an Axiovert 135 microscope (Zeiss) equipped with an AxioCam camera. Dead cells were excluded by PI staining. Apoptosis was also measured by a single stranded DNA (SS DNA) apoptosis ELISA kit (chemicon). Western Blot analysis Synoviolin expression was examined by Western blot using antibodies against synoviolin (Abgent) [3] or b-actin (Santa Cruz, Cell Signaling Laboratories) as described previously [45]. Membranes were stripped and serially reprobed with antibodies against synoviolin and b-actin used as a loading control. Real-time RT-PCR Analysis 1 mg of RNA extracted using TRIzol reagent (Gibco BRL) was reverse transcribed using Thermoscript RT-PCR system (Invitro- gen, California, USA) and PCR amplification performed on a LightCycler (Roche) using Fast-StartTM DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals) [3]. Primer-specific nucleotide sequences for c-fos (Search-LC GmbH, Heidelberg, Germany), c-jun (Search-LC GmbH, Heidelberg, PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13416