Figure 6
P2X7 activation mediates dASC cell death. (a) After 1 h incubation with 5 mM of ATP, cells acquired a rounded morphology typical of dying cells. Cell death was prevented by preincubation with the specific P2X7 antagonist AZ 10606120 dihydrochloride (300 nM), as shown by bright field images. NT, non-treated controls. (b) LDH assay was used to measure cytotoxicity following ATP (1–10 mM) treatments, and a significant increase of cell death was observed only at 5 and 10 mM ATP. (c) AZ 10606120 dihydrochloride significantly reduced the ATP-induced cytotoxicity to levels comparable to the controls. Data were normalised to the LDH levels of Triton-X lysates and expressed as percentage of cytotoxicity±S.E.M. (d) An MTS assay was performed to measure the cell viability ATP treatment significantly reduced cell viability compared with the NT controls. Pretreatment with AZ 10606120 dihydrochloride prevented the ATP-dependent decrease in cell survival restoring cell viability to levels comparable to NT samples. (e) P2X7-dependent ATP-induced cell death was further confirmed with EthD-1 staining. Following ATP treatments, the number of death cell stained by EthD-1 was significantly increased. This was prevented by incubation with the AZ 10606120 dihydrochloride compound. For all assays, statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, n=6, **P<0.01, ***P<0.001 and ****P<0.0001)
