IL-17 and Synoviocyte Survival reduction compared to sictl treated samples, n = 5, mean 6 SEM of duplicate experiments. * P,0.05, compared to sictl nucleofected RA FLS. (B), RA FLS were nucleofected as above then treated with SNP 0.1–1 mM overnight and apoptosis measured by the SS DNA apoptosis assay. Results are expressed as fold induction in apoptosis, n = 3 from 3 separate RA donors, mean 6 SEM of duplicate experiments. * P,0.05, compared to sictl nucleofected RA FLS. (C), RA FLS were nucleofected as above, serum starved overnight then pretreated with 100 ng/ml IL-17A for 2 h then cotreated with SNP 0.1–1 mM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis, n = 3 from 3 separate RA donors, mean 6 SEM of duplicate experiments. * P,0.05, compared to sictl nucleofected RA FLS. { P,0.05 compared to SNP treatment in S-08 nucleofected RA FLS. doi:10.1371/journal.pone.0013416.g004 Methods Ethics statement The use of patient biological samples and this study was reviewed and approved by the local institutional review committee of the hospital of Lyon, and patients gave informed written consent. Isolation and culture of RA FLS RA FLS were obtained from synovial tissue obtained from RA patients undergoing joint surgery who fulfilled the American College of Rheumatology criteria for RA [41]. Osteoarthritis (OA) FLS or dermal fibroblasts were obtained from the joint or the skin of patients with OA undergoing joint surgery; these cells were used Figure 5. Effect of IL-17 and TNF on RA FLS synoviolin expression and apoptosis. RA FLS were stimulated with IL-17A (50 or 100 ng/ml) and/or TNF (1 or 10 ng/ml) for 24 h and synoviolin mRNA expression measured by real-time RT-PCR (A). The results based on a ratio of synoviolin/b- actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean 6 SEM. * P,0.05 compared to no treatment control. { P,0.05 compared to TNF treated samples. RA FLS were pretreated with IL-17 100 ng/ml and TNF 10 ng/ml for 2 h then cotreated with SNP 0.5 mM (B, left panel) or 0.1 mM (B, right panel) for 24 h and apoptosis measured by SS DNA apoptosis kit. The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean 6 SEM of duplicate experiments from 3 different RA donors. * P,0.05 compared to SNP treatment in S-08 nucleofected RA FLS. doi:10.1371/journal.pone.0013416.g005 PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13416