Fig. 4.

p38 MAPK and caspase activation, but not ROS formation, mediates P2X7-induced apoptosis in MEL cells. a, c and d MEL cells in complete culture medium were pre-incubated at 37 °C for 30 min a in the absence (control) or presence of 10 mM NAC, in the presence of c DMSO, 20 μM SB202190 or 20 μM SB203580, or d DMSO or 20 μM Z-VAD-FMK. Cells were then incubated in the absence (basal) or presence of 1 mM ATP at 37 °C for 24 h. Cells were harvested and labelled with Annexin-V-FLUOS and 7AAD, and the percentage of Annexin-V⁺/7AAD⁻ (early apoptotic; left) or Annexin-V⁺/7AAD⁺ (late apoptotic; right) cells was analysed by flow cytometry. b H₂DCFDA-loaded MEL cells in NaCl medium or complete culture medium (CC) were incubated in the absence (basal) or presence of 1 mM ATP at 37 °C for 15 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. e MEL cells in NaCl medium were pre-incubated at 37 °C for 30 min in the presence of DMSO or 20 μM SB202190 (left), DMSO or 20 μM SB203580 (centre) or DMSO or 20 μM Z-VAD-FMK (right). Ethidium⁺ (25 μM) was added and cells were incubated in the absence (basal) or presence of 1 mM ATP at 37 °C for 15 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the MFI of ethidium⁺ uptake (pore formation) was analysed by flow cytometry. a–e Results are mean ± SD (n = 3); *P < 0.05 compared with corresponding basal; **P < 0.01 compared with corresponding basal; †P < 0.05 compared with ATP alone; ††P < 0.01 compared with ATP alone