Inhibition of the P2X7 signaling pathway does not affect the cytokine-induced release of microvesicles (MVs) from CHME-5 cells. Quantification of MV release by flow cytometry (A,B,D,E,G,H) and western blotting for flotillin-1 (C,F,I) in the presence or absence of multiple inhibitors of the P2X7 signaling pathway. Data on the graphs are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). (A–C) Cells were treated with cytokines (20 ng ml−1) for 24 h in the presence or absence of oxidized ATP (oxATP) (200 µM) (A,C) or brilliant blue G (BBG) (100 nM) (B). ATP treatments (1 mM, 30 min) ± inhibitors (24 h pre-treatments) were also performed. MVs were then collected and analyzed by flow cytometry and western blot. Unpaired two-tailed Student’s t-test was used for statistical analyses (***p: 0.0006/p: 0.0002). No significant difference of cytokine-induced MV release was found in the presence of oxATP and BBG that, however, reduce the MV release induced by ATP. (D–F) The same as in (A–C) but in the presence of two pannexin-1 inhibitors: probenecid (prob) (5 mM) (D,F) and the blocker peptide 10Panx1 (300 µM) (E). No significant difference of cytokine- and ATP-induced MV release was found in the presence of probenecid and 10Panx1. (G–I) Imipramine (imipr) (10 µM) (G,I) and siramesine (sir) (8 µM) (H) were used as acid sphingomyelinase inhibitors. Unpaired two-tailed Student’s t-test was used for statistical analyses (*p: 0.0342/***p: 0.0004). No significant difference of cytokine-induced MV release was found in the presence of imipramine and siramesine, whereas they significantly reduce the release mediated by ATP. In all experiments inhibitors were applied 1 h before cytokine treatment, or for 24 h before ATP treatment, and maintained thereafter, to allow comparison of the effects of the inhibitors on ATP and cytokine-mediated MV production. All the western blots shown are representative of two independent experiments. Full-length blots are presented in Figure S4 in Supplementary Material.