Apoptosis was determined by quantifying mono and oligonucleosomal DNA using the Cell Death Detection ELISA kit as previously described. Apoptosis was confirmed by Hoechst staining and measure of acti vated caspase 3/7 by the Caspase Glo 3/7 assay. RA FLS were cultured either on 24 well plates or 96 well plates, treated for one hour with 1 uM Wort or 10 uM LY and then incubated for 12 hours with 1 ug/ml of human anti Fas. After incubation, plates were stained with 10 ug/ml Hoechst 33258, fixed with 4% paraformaldehyde and the cells were examined by fluorescence microscopy. For activated caspase 3/7 analysis, cells were incubated for one hour with reconstituted Caspase 3/7 Glo reagent and then, the lumi nescence signal generated after cleavage of DEVD amino luciferin substrate by caspase 3/7, was measured using a Fluostar OPTIMA microplate reader.

Western blot analysis After siRNA transfections, RA FLS were cul tured in six well plates, treated for one hour with 1 uM Wort and then stimulated with human anti Fas 1 ug/ml for 3 or 12 hours. Cells were washed twice with ice cold PBS, and protein was extracted using lysis buffer, 100 mM NaF, 1 mM Na3VO4, 10 ug/ml aprotinin, 10 ug/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The protein concentrations in the extracts were determined using the Qubit fluorometer according to the manufacturers protocol. Whole cell lysates were fraction ated by Tris glycine buffered 10% SDS PAGE and trans ferred to polyvinylidene fluoride membrane. The membranes were blocked with Tris buffered saline and 0.

1% Tween 20 containing 5% non fat milk for two hours at room temperature, followed by incubation with antibody to phospho Akt, Akt, Bid, Caspase 9 or B actin overnight at 4 C. After washing with TBST, the membrane was incubated with horseradish peroxidase con jugated secondary antibody. Statistical analysis Differences between experimental groups were assessed by Wilcoxon matched pairs test. P values less than 0. 05 were considered significant. Results Regulation of Fas mediated apoptosis in RA FLS by Akt RA FLS from six patients were pre treated for one hour with Wort or LY, and stimulated thereafter with Fas anti body for 12 hours. Apoptosis of RA FLS was determined by analysis of nucleosomal release, Hoechst staining and activated caspase 3/7 measurement. As a positive control we analysed the GSK-3 nucleosomal release after anti Fas stimula tion in Jurkat cells. Mean DO492 nm was 0. 93 versus a mean of 0. 13 observed in the six RA FLS, confirming the relative resistance of these latter cells to Fas induced apop tosis. In RA FLS, anti Fas stimulation induced significant apoptosis compared with the basal situation.