IL-17 and Synoviocyte Survival time RT-PCR, normalised to GAPDH and expressed as percentage inhibition compared to sictl siRNA (B). * P,0.05, compared to sictl nucleofected RA FLS. RA FLS were nucleofected as above, serum starved overnight then pretreated with 50 ng/ml IL-17A for 2 h then cotreated with SNP 0.1 mM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis (C), n = 3 from 3 separate RA donors, mean 6 SEM of duplicate experiments. * P,0.05, compared to sictl nucleofected RA FLS. RA FLS were nucleofected as above serum starved overnight then treated with 50 ng/ml IL-17A and (D) synoviolin levels analysed by real-time RT-PCR. Synoviolin mRNA level was normalized to GAPDH and expressed as fold increase over sictl siRNA treated samples, n = 5, mean 6 SEM of duplicate experiments. * P,0.05, compared to sictl nucleofected RA FLS. { P,0.05 compared to IL-17 sictl treated samples. RA FLS were pretreated with 2 mg/ml of anti-IL-17RA or anti-IL-17RC Abs or isotype control goat Abs for 2 h then cotreated with IL-17 50 ng/ml for 24 h and synoviolin expression measured by real-time RT-PCR (E) , n = 3, mean 6 SEM. * P,0.05, compared to untreated control. { P,0.05 compared to IL-17 treated samples. doi:10.1371/journal.pone.0013416.g003 Figure 4. Effect of synoviolin knockdown on apoptosis in RA FLS. RA FLS were nucleofected (amaxa) for 24 h with 0.5 mg of 4 synoviolin siRNA duplexes (S-01 to S-04), synoviolin smartpool siRNA duplex or siCONTROL siRNA (sictl) and synoviolin expression analysed by real-time RT-PCR (A, left panel) or Western Blot (A, right panel). Synoviolin mRNA was normalized to GAPDH and results expressed as the percentage fold PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13416