Panx1 blockage decreases ATP release from N2a cells and blocking P2 receptors reduces cell proliferation. (A) Panx1 is endogenously expressed in N2a cells, as demonstrated by RT-PCR (i) and western blot analysis (ii). To the left of the western blot, the asterisk (*) above the arrow denotes high-molecular weight species likely representing various Panx1 oligomeric forms, while the asterisk below the arrow denotes low molecular weight Panx1 cleavage products consistent in size with those reported in the literature . (B) ATP release from N2a cells was stimulated upon treatment with 20 mM KCl buffer compared to control (5.33 mM) and 0 mM KCl conditions ((**) P < 0.01 for one-way ANOVA with post-hoc Tukey’s multiple comparison test, N = 3). (C) Blockage of Panx1 channels with 0.5 mM probenecid significantly decreases stimulated ATP release from N2a cells compared to controls (60.50 ± 7.913% and 99.17 ± 6.831%, respectively, N = 5, P = 0.0159 for non-parametric t test). (D) N2a cell numbers were quantified at 0, 24 and 48 hours for cells treated with 30 μM PPADS, 1 mM probenecid, or vehicle control. PPADS and probenecid treatments significantly reduced N2a numbers at 24 and 48 hours ((*) P < 0.05 and (***) P <0.001 for one-way ANOVA with post-hoc Tukey’s multiple comparison test, N = 3). ANOVA, analysis of variance; Panx, pannexin1, PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid.