IC₅₀ or EC₅₀ values for P2X7R agonists or antagonists using different methods.

<p>*IC₅₀ for natively expressed mP2X7R in J774.G8 cells.</p><p>**N.D. = Not determined</p><p>IC₅₀ or EC₅₀ values for P2X7R agonists or antagonists using different methods.</p

Pannexin-1 Blockage Assay in U937 Cells.

<p>U937 cells were plated in opaque 96-well plates following the subsequent treatments: a) Cells plus PI [50 nM]; b) cells plus Triton X-100 [0.1%] plus PI [50 nM]; c) cells plus ATP [5 mM] plus PI [50 nM]; d) cells plus BBG [100 nM] plus PI [50 nM]; e) cells plus Carbenoxolone [100 μM] plus PI [50 nM]; f) cells plus Mefloquine [1 μM] plus PI [50 nM]; g) cells plus AZ11645373 [10 μM] plus PI [50 nM]; h) cells plus BBG [100 nM] plus ATP [5 mM] plus PI [50 nM]; i) cells plus Carbenoxolone [100 μM] plus PI [50 nM] plus ATP [5 mM]; j) Cell plus Mefloquine [1 μM] plus ATP [5 mM] plus PI [50 nM]; l) cells plus AZ11645373 [10 μM] plus PI [50 nM] plus ATP [5 mM]. Date represents mean ± S.D of three independent experiments in triplicate. The result was analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p

OATP dose-response curve.

<p>J774.G8 cells were plated in opaque 96-well plates and treated with various concentrations of OATP from 0.048 to 400 μM. The data represent the means ± S.D. of three independent experiments in triplicate. The results were analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p

ATP dose-response curve.

<p>J774.G8 cells were plated in opaque 96-well plates and treated with various concentrations of ATP from 0.321 to 25 mM in combination with PI [50 nM]. The data represent the means ± S.D. of three independent experiments in triplicate. The results were analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p

BBG dose-response curve.

<p>J774.G8 cells were plated in opaque 96-well plates and treated with various concentrations of BBG from 0.048 to 100 μM in combination with PI [50 nM] and ATP [5 mM]. The data represent the means ± S.D. of three independent experiments in triplicate. The results were analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p

MTT cytotoxicity assay.

<p>J774.G8 cells that were treated for 15 minutes with five concentrations of ATP were analyzed. (a) Untreated cells, (b) cells plus Triton X-100 [0.1%], (c) cells plus ATP [5 mM], (d) cells plus ATP [10 mM], (e) cells plus ATP [15 mM], (f) cells plus ATP [20 mM] and (g) cells plus ATP [25 mM]. The data represent the means ± S.D. of three independent experiments in triplicate. The results were analyzed using ANOVA, followed by Tukey's post-hoc test; * p<0.05.</p

Anionic dye uptake.

<p>J774.G8 cells were plated in opaque 96-well plates after the following treatments: a) Cells plus LY [3 mM], b) cells plus ATP [5 mM] plus LY [3 mM], c) cells plus Triton X-100 [0.1%] plus LY [3 mM], d) cells plus BBG [100 nM] plus ATP [5 mM] plus LY [3 mM] and e) cells plus OATP [300 μM] plus ATP [5 mM] plus LY [3 mM]. The data represent the means ± S.D. of three independent experiments in triplicate. The results were analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p

Evaluation of the inhibitory activity of BBG on P2X7 using fluorescence microscopy.

<p>J774.G8 cells were treated as follows: (a) Control (J774 plus PI [50 nM]), (b) permeabilization control (Triton-X 100 [0.1%]), (c) control cells plus ATP [5 mM], (d) cells treated with BBG [100 nM] plus ATP [5 mM] and (e) cells treated with OATP [300 μM] plus ATP [5 mM]. The profiles are representative of three to seven independent experiments that were performed in triplicate. The images were captured using a Nikon digital camera system with a total magnification of 43.8X.</p

Pannexin-1 Blockage Assay in J774.G8 Cells.

<p>J774.G8 cells were plated in opaque 96-well plates following the subsequent treatments: a) Cells plus PI [50 nM]; b) cells plus Triton X-100 [0.1%] plus PI [50 nM]; c) cells plus ATP [5 mM] plus PI [50 nM]; d) cells plus BBG [100 nM] plus PI [50 nM]; e) cells plus Carbenoxolone [100 μM] plus PI [50 nM]; f) cells plus Mefloquine [1 μM] plus PI [50 nM]; g) cells plus AZ11645373 [100 μM] plus PI [50 nM]; h) cells plus BBG [100 nM] plus ATP [5 mM] plus PI [50 nM]; i) cells plus Carbenoxolone [100 μM] plus PI [50 nM] plus ATP [5 mM]; j) cells plus Mefloquine [1 μM] plus ATP [5 mM] plus PI [50 nM]; l) cells plus AZ11645373 [100 μM] plus PI [50 nM] plus ATP [5 mM]. Date represents mean ± S.D of three independent experiments in triplicate. The result was analyzed using the Kruskal-Wallis, followed by Dunns post-hoc analysis; * p<0.05.</p