Figure 7
Celecoxib does not induce caspase activation in rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs). (a) FLSs were stimulated for 2 hours with celecoxib at indicated concentrations or for 4 hours with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (0.5 nM) as positive control. Cell lysates were analyzed by immunoblot for caspase 3 expression. The same blot was stripped and reprobed with a mouse anti-human β-actin antibody to confirm equal loading. One representative immunoblot is shown. (b) RA FLSs were stimulated for indicated time points with 60 μM celecoxib or with TRAIL (0.5 nM) as positive control, and caspase 3 activity was measured using Ac-DEVD-AMC protease assay. Caspase 3 activity is expressed as fold increase to unstimulated cells (NS) and is represented as the mean ± standard error of the mean (SEM) of different experiments using RA FLSs from three different patients. (c) RA FLSs were stimulated for indicated time points with celecoxib at indicated concentrations or with TRAIL (0.5 nM) as positive control. Cell lysates were analyzed by immunoblot for poly(ADP-ribose) polymerase (PARP) and caspase 8 and 9 expression. One representative immunoblot is shown. (d) RA FLSs were stimulated for 12 hours with celecoxib at indicated concentrations or with TRAIL (0.5 nM) as positive control, and DNA fragmentation was measured using the Cell Death Detection ELISAPLUS kit. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as the ratio of the absorbance of the sample cells to the absorbance of control cells and is shown as the mean ± SEM from three experiments performed in duplicate.