Fig. 3

TNFα-induced PARP-1 activation requires PLC, Ca²⁺ influx, and activation of MAPK-ERK kinase. a Astrocyte cultures were immunostained for PAR after 60-min incubations with 15 ng/ml TNFα. PAR formation was prevented in calcium-free medium (containing 500 μM EGTA) and by the calcium chelator BAPTA-AM (2 μM), but not by pre-treatment with thapsigargin (2 μM for 15 min). PAR formation was also prevented by the PC-PLC inhibitor, D609 (10 μM), and by the MAPK-ERK inhibitor, PD98059 (10 μM). The PI-PLC inhibitor U-73112 (10 μM) did not block PAR formation. An absent signal in cultures treated with the PARP inhibitor DPQ (25 μM) confirms the specificity of the immunostaining. Representative of n = 4. b PAR immunostaining in microglia shows the same pattern as observed in astrocytes. Studies using EGTA were not possible with microglia cultures due to rapid cell detachment. c Phase contrast images of same microglial files shown in b. Microglial morphological transformation induced by TNFα is absent under the conditions in which PAR formation was blocked. Scale bars, 50 μm. d Quantification of PAR positive cells (avarage number per field in confluent astoglia cultures; percent of positive cells in microglia cultures). (*p < 0.05, n = 3–4)