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

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

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

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

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

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

Modulation of migration by nucleotides in a pleurisy model.

<p>Rats were sensitized with OVA in the first day. On day 14 all animals were treated or not with agonists and antagonists of P2R, and after 1h animals were of challenged. After 24h, cells in the pleural cavity was collected and counted for total cells (Fig 5A) and eosinophils (Fig 5B). *** p<0. 05 when saline group was compared to other treatments; +++ p<0.01 when UTP group was compared to ATPγS group; ++ p<0.05 when UTP group was compared to ATP group; # p<0.05 when ATP group was compared to Suramin group; ### p<0.01 when UTP group was compared to Suramin group.This experiment was realized for 3 times with 6 animals per group.</p