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 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. Treatment with Wort or LY did not induce cell death by themselves, whereas when combined with anti Fas they significantly increased the apoptotic rate when compared with anti Fas alone, as has been shown in our previous work. Connection between the intrinsic and extrinsic apoptotic pathways in RA FLS There is some indication that RA FLS are type II cells in relation to apoptosis because Bid was cleaved after anti Fas stimulation. Cilengitide We have confirmed these results showing that after incubation with anti Fas the detectable full Bid protein is significantly decreased in all RA FLS lines analy sed. Furthermore, we wanted to know whether the cleavage of Bid is essential for apoptosis in RA FLS. To this end, Bid was suppressed in RA FLS from five different patients and the efficiency of Bid silencing is shown in Fig ures 2b and 2c. Interestingly, suppression of Bid completely abrogated Fas induced apoptosis.