Figure 4
TNF-a must activate NF-kB signaling to protect macrophages from MTX-induced apoptosis. A. PAR and BAY (10 μM) prevent TNF-a-induced NF-kB activity in RAW264.7 cells, as measured by activity of the pNF-kB-TA-Luc reporter. Inhibitors were introduced 30 minutes before TNF-a and luciferase activity was measured nine hours later. Results are mean ± SEM of at least four independent experiments. B-F. PAR and BAY (10 mM or 7.5 mM) prevent TNF-a from rescuing MTX-treated RAW264.7 cells (B and C) and BMDM (D-F) from apoptosis. Cells were exposed to PAR (B, D, and E) or BAY (C and F) for 30 minutes before TNF-a stimulation for three hours, after which MTX was introduced. Annexin-V binding and/or caspase-3 activity was measured 6 hours later in RAW264.7 cells and 24 hours later in BMDM. Results are mean ± SEM of at least three independent experiments in each case. Normalised data are shown for BAY experiments. * = P < 0.05; ** = P < 0.01.