Journal: Journal of Cardiovascular Development and Disease

Article Title: Role of P2X7 Receptor on Hypoxia-Induced Vascular Endothelial Growth Factor Gene Expression in H9c2 Rat Cardiomyocytes

doi: 10.3390/jcdd12110438

Figure Lengend Snippet: Effect of A740003 and Echinomycin treatment on cytotoxic activity in H9c2 cells exposed to hypoxia. LDH activity in H9c2 cells exposed to 6 h of hypoxia, 5 μM A740003 + hypoxia and 1nM Echinomycin + hypoxia. Data are presented as mean ± SEM ( n ≥ 3). One-way analysis of variance (ANOVA) test was used followed by Tukey’s multiple comparisons test. **** p < 0.0001 compared to normoxia control group.

Article Snippet: A740003 and Echinomycin were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Activity Assay, Control

Journal: Journal of Cardiovascular Development and Disease

Article Title: Role of P2X7 Receptor on Hypoxia-Induced Vascular Endothelial Growth Factor Gene Expression in H9c2 Rat Cardiomyocytes

doi: 10.3390/jcdd12110438

Figure Lengend Snippet: Effect of A740003 and Echinomycin treatment on VEGF level in H9c2 cells exposed to hypoxia. VEGF levels in H9c2 cardiomyocytes exposed to 6 h of hypoxia, 5 μM A740003 + hypoxia and 1nM Echinomycin + hypoxia. Data are presented as mean ± SEM ( n ≥ 3). One-way analysis of variance (ANOVA) test was used followed by Tukey’s multiple comparisons test. *** p < 0.001 compared to normoxia control group; * p < 0.05 and ** p < 0.01 compared to hypoxia group.

Article Snippet: A740003 and Echinomycin were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: Journal of Cardiovascular Development and Disease

Article Title: Role of P2X7 Receptor on Hypoxia-Induced Vascular Endothelial Growth Factor Gene Expression in H9c2 Rat Cardiomyocytes

doi: 10.3390/jcdd12110438

Figure Lengend Snippet: Effect of A740003 and Echinomycin treatment on HIF-1α and VEGF mRNA expression level in H9c2 cells exposed to hypoxia. ( a ) HIF-1α and ( b ) VEGF mRNA expression levels in H9c2 cardiomyocytes exposed to 6 h of hypoxia, 5 μM A740003 + hypoxia and 1nM Echinomycin + hypoxia. Data are presented as mean ± SEM ( n ≥ 3). One-way analysis of variance (ANOVA) test was used followed by Tukey’s multiple comparisons test. *** p < 0.001 and **** p < 0.0001 compared to normoxia control group; *** p < 0.001 and **** p < 0.0001 compared to hypoxia group.

Article Snippet: A740003 and Echinomycin were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Control

Journal: Journal of Cardiovascular Development and Disease

Article Title: Role of P2X7 Receptor on Hypoxia-Induced Vascular Endothelial Growth Factor Gene Expression in H9c2 Rat Cardiomyocytes

doi: 10.3390/jcdd12110438

Figure Lengend Snippet: Schematic representation of the role of P2X7Rs in pro-angiogenic signaling in response to hypoxic cardiomyocytes. ( a ) Hypoxic cardiomyocytes activate P2X7R, which stimulates cardiac pro-angiogenic signaling through HIF-1α/VEGF. ( b ) A740003, a P2X7R antagonist, inhibits pro-angiogenic signaling in hypoxic cardiomyocytes by inhibiting the HIF-1α/VEGF pathway. Abbreviations: eATP, extracellular adenosine 5-triphosphate; iATP, intracellular adenosine 5-triphosphate; HIF-1α, Hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor. Blue circles represent ATP, while the red circle represents the P2X7R antagonist A740003. (Created with BioRender.com ).

Article Snippet: A740003 and Echinomycin were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: Prophylactic systemic P2X7 receptor blockade prevents experimental colitis.

doi: 10.1016/j.bbadis.2013.10.012

Figure Lengend Snippet: Fig. 8. Effect of P2X7-R blockade with A740003 on the colonoscopic parameters and histological parameters in the colitis model. Colonoscopic imaging was obtained after colitis induction at day 7. In control experiments, colonoscopy was performed following intra-rectal saline enemas, and intraperitoneal administration of vehicle in TNBS-induced animals. Animals treated with intraperitoneal (IP) A740003, showed significantly less inflammation as assessed by colonoscopy, in contrast to vehicle-treated animals (p = 0.005). Values are mean ± S.E.M. of 5–6 animals per group. Significant values are presented (A). Paraffin sections were stained with HE. Intraperitoneal (IP)-A740003 was administered at 16 mg/kg at day –1. In control experi- ments, histological evaluation was performed following intraperitoneal administration of vehicle, and intra-rectal saline enemas. Colonic samples were scored according to histological pa- rameters described in the Materials and methods section. Animals treated with IP-A740003 showed significantly less inflammation measured by histologic parameters compared to vehicle-treated animals (p = 0.034). Values are mean ± S.E.M. of 5–6 animals per group. Significant values are presented (B). Length bars represent 20 μm.

Article Snippet: The P2X7-R blockers utilized in this work were the brilliant blue G (BBG) (Sigma-Aldrich, Saint Louis, MO, USA) and A740003 [N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl) acetamide] (Tocris Bioscience, Bristol, UK) [35].

Techniques: Imaging, Control, Saline, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome

doi: 10.1016/j.jcmgh.2025.101718

Figure Lengend Snippet: The colocalization of P2X7 ( red ) with α-SMA ( green ) was significantly greater in the samples from the patients with CD and UC than in those from the noninflamed controls (NL). ( A ). Nuclei are stained with DAPI ( blue ). The micrograph panels are representative of 8 control, 8 CD, and 8 UC mucosal samples (original magnification, ×1000). The scale bars represent 50 μm. The percentages of double-positive cells in the colonic lamina propria are individually represented ( A ). Intracellular calcium ([Ca 2+ ] i) levels were assessed in CCD-18Co cells after plating at 1 × 10 5 cells per well on 15 mm coverslips. Upon exposure to ATP, a transient rise in calcium is observed, returning to basal levels after ∼2 minutes. However, pre-incubation with LPS enhanced the calcium influx peak, followed by a slower return to baseline after ∼3 minutes ( B and C ). The micrograph panel is representative of 3 experiments for each condition ( D ). Next, pretreatment with the P2X7 receptor antagonist A740003 strongly inhibited the calcium influx observed in ATP-incubated cells ( E and F ). The micrograph panel is representative of 3 experiments for each condition ( G ). In another set of experiments, the relative intensity of P2X7 in the CCD-18Co cells subjected to different treatments was determined via confocal microscopy. Compared with the controls, P2X7 expression significantly increased after treatment with ATP (0.5 mM or 2 mM), LPS, IL-1b, with or without ATP (2 mM). In contrast, expression decreased after KN62 + ATP (2 mM) compared with LPS + ATP (2 mM). The micrograph panel is representative of 7 experiments for each condition. The scale bars represent 20 mm ( H ). The effects of different stimuli that activate P2X7 receptors on CCD-18Co migration were analyzed by light microscopy. Panels show images of migration from 0 to 24 hours into scratch sites following growth in medium containing vehicle or other stimuli. Compared with the KN62-treated cells, the BzATP-treated cells previously exposed to LPS, IL-1β, or TGF-β exhibited significantly increased migration. The scale bars represent 20 μm. The micrograph panel is representative of 4 experiments for each condition ( I ). Individual values are presented, along with their means and SDs. For comparing the means of 3 or more groups, the analyses were performed using Welch’s ANOVA test, followed by multiple comparisons using Dunnett’s T3 test. Paired samples t -test was used to determine differences in calcium influx experiments. Significant values are highlighted.

Article Snippet: Cells were continuously perfused with a fluorimetry solution and stimulated with 2 mM ATP (A2383, Sigma-Aldrich) or 100nM A740003 (3701, TOCRIS Bioscience).

Techniques: Staining, Control, Incubation, Confocal Microscopy, Expressing, Migration, Light Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome

doi: 10.1016/j.jcmgh.2025.101718

Figure Lengend Snippet: At the end of week 8, a significant increase in mortality was observed in the DSS-induced P2X7R +/+ mice compared with the non-exposed control mice ( A ). Compared with those in the control groups, the weight of the mice in the DSS groups did not increase over time ( B ). After the third DSS induction week, colonoscopy images were obtained 2 cm from the anus, and eUBM and histological axial analysis were performed ( C ). The colonoscopy ( D ) and the histological ( E ) scores were higher in the DSS-induced P2X7 +/+ than in the P2X7 +/+ control (CTL) group, the P2X7 -/- mice, and the DSS-induced P2X7 +/+ treated with A740003. The analysis was performed using Welch’s ANOVA, with multiple comparisons conducted using Dunnett’s T3 test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. Individual values are shown, along with the means and SDs from 2 independent experiments (5–7 per group).

Article Snippet: Cells were continuously perfused with a fluorimetry solution and stimulated with 2 mM ATP (A2383, Sigma-Aldrich) or 100nM A740003 (3701, TOCRIS Bioscience).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome

doi: 10.1016/j.jcmgh.2025.101718

Figure Lengend Snippet: eUBM was performed on anesthetized mice after the third week of DSS induction. Colon stiffness was assessed with an intrarectal injection of 0.5 mL of saline solution at times t0, t1, and t2 (10 seconds in total) during ultrasound image acquisition ( A ). The increase in the transverse colon section area was analyzed in the experimental groups. The DSS-induced P2X7 +/+ mice presented a lower increase in the transversal section area than the control, A740003-treated DSS-induced P2X7 +/+ , and DSS-induced P2X7 -/- mice ( B ). The analysis was performed using Wilcoxon’s signed-rank test. The horizontal bars represent the medians, and the boxes represent the 25th and 75th percentiles. The data are representative of 2 independent experiments (4–5 per group). Collagen production in colon homogenates from the P2X7 -/- and P2X7 +/+ explants treated with A740003 was lower than in the DSS-induced P2X7 +/+ explants ( C ). Procollagen production in colon homogenates from the P2X7 -/- and P2X7 +/+ explants treated with A740003 was lower than in the DSS-induced P2X7 +/+ explants ( D ). The analysis was performed using Welch’s ANOVA, with multiple comparisons conducted using Dunnett’s T3 test. Individual values are presented, along with the means and SDs of 2 independent experiments (5–7 per group).

Article Snippet: Cells were continuously perfused with a fluorimetry solution and stimulated with 2 mM ATP (A2383, Sigma-Aldrich) or 100nM A740003 (3701, TOCRIS Bioscience).

Techniques: Injection, Saline, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome

doi: 10.1016/j.jcmgh.2025.101718

Figure Lengend Snippet: Cytokine levels were assessed in the supernatants of colon explants cultured for 24 hours. Genetic inactivation of P2X7 resulted in decreased levels of IL-1β ( A ), TGF-β ( B ), IL-10 ( C ), IFN-γ ( D ), TNF-α ( E ), IL-17 ( F ), and IL-6 ( G ) compared with those in P2X7 +/+ mice. A740003 was used as a selective inhibitor of P2X7 for treating DSS-induced P2X7 +/+ mice. The analysis was performed using Welch’s ANOVA test, followed by multiple comparisons using Dunnett’s T3 test. Individual values are presented, along with the means and SDs of 2 independent experiments (n = 5 per group).

Article Snippet: Cells were continuously perfused with a fluorimetry solution and stimulated with 2 mM ATP (A2383, Sigma-Aldrich) or 100nM A740003 (3701, TOCRIS Bioscience).

Techniques: Cell Culture

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome

doi: 10.1016/j.jcmgh.2025.101718

Figure Lengend Snippet: Paraffin sections of colon samples stained with indirect immunoperoxidase were analyzed via a computerized image analysis system. P2X7 receptor blockade via genetic inactivation significantly attenuated the increase in the number of NF-κB ( A ), phosphorylated ERK ( B ), caspase-1 ( C ), and NLRP-3 ( D ) -positive cells observed in DSS-induced P2X7 +/+ mice. Using the oil-immersion technique, most positive cells for NF-κB, p-ERK, and caspase-1 in slides from DSS-induced P2X7 +/+ mice showed intense nuclear staining, whereas in the other groups, staining was markedly weaker and mostly cytosolic ( A–C ). For NLRP3, positive cells in slides from DSS-induced P2X7 +/+ mice showed intense, diffuse cytosolic staining, whereas in the other groups, staining was markedly weaker ( D ). In contrast, P2X7 receptor blockade decreased the number of PPAR-γ-positive cells ( E ). A740003 was used as a selective inhibitor of P2X7 for treating DSS-induced P2X7 +/+ mice. The scale bars represent 50 μm. The analysis was performed using Welch’s ANOVA, with multiple comparisons conducted using Dunnett’s T3 test. Individual values are shown, along with the means and SDs of 3 independent experiments (n = 7 per group).

Article Snippet: Cells were continuously perfused with a fluorimetry solution and stimulated with 2 mM ATP (A2383, Sigma-Aldrich) or 100nM A740003 (3701, TOCRIS Bioscience).

Techniques: Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 inhibited the activation of NLRP3 inflammasome and phosphorylation of IKB α and decreased the expression of P2X7R in ARPE-19 cells exposed to ox-LDL for 24 hours. ELISA results showed that A740003 pretreatment decreased the secretion of inflammatory cytokines including IL-1 β (to 2.831 ± 0.1162) (a) and IL-18 (to 1.585 ± 0.2329) (b) significantly. Besides, compared to the vehicle-treated group, A740003 pretreatment significantly inhibited NLRP3 (56.73% ± 0.3908) (c) and P2X7R (51.30% ± 0.1196) (d) at mRNA levels in ARPE-19 cells. It indicated that A740003 inhibited the overexpression of P2X7R and NLRP3 which was induced by ox-LDL. Western blot showed that A740003 downregulated the protein levels of NLRP3 (64.92% ± 0.1264) (e, g), pro-Caspase-1 (68.26% ± 0.02456) (f, j), Caspase-1 (65.97% ± 0.01661) (f, k), and P2X7R (60.55% ± 0.09754) (e, h) and the phosphorylation of IKB α (79.34% ± 0.01995) (e, i) significantly compared to the vehicle-treated group. Symbols: +: with; −: without. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction.

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 suppressed the upregulation of angiogenic growth factors in ARPE-19 cells induced by ox-LDL incubation for 48 hours. The mRNA levels of VEGF and HIF-1 α were detected by qPCR. Compared to the vehicle-treated group, A740003 pretreatment significantly suppressed the mRNAs of VEGF (38.12% ± 0.2505) (a) and HIF-1 α (30.28% ± 0.2455) (b) in ARPE-19 cells. Besides, the protein levels of VEGF and HIF-1 α were detected by Western blot. A740003 pretreatment also decreased the overexpression of VEGF (51.03% ± 0.01724) (c, d) and HIF-1 α (66.43% ± 0.03408) (c, e) compared to vehicle-treated cells. Symbols: +: with; −: without. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction.

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Incubation, Western Blot, Over Expression

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 inhibited the ROS production in ARPE-19 cells induced by ox-LDL. The fluorescence of DCFH-DA increased robustly in ARPE-19 cells incubated with ox-LDL (a). The average optical density of ROS in the ox-LDL group was obviously higher than that in the control group (b). However, A740003 pretreatment could decrease the fluorescence of DCFH-DA in ARPE-19 cells induced by ox-LDL (a, b). The overproduction of ROS induced by ox-LDL was suppressed by A740003. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction.

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Fluorescence, Incubation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 inhibited the activation of NLRP3 inflammasome and phosphorylation of IKB α and decreased the overexpression of P2X7R in C57BL/6 mouse retinas injected with ox-LDL. The qPCR showed that compared to the vehicle-treated group, intraperitoneal injection of A740003 reduced the mRNA expression of NLRP3 (29.03% ± 0.05369) (a), Caspase-1 (39.79% ± 0.3646) (b), and P2X7R (34.73% ± 0.09999) (c) in C57BL/6 mouse retina obviously. Moreover, Western blot showed that protein levels of NLRP3 (55.03% ± 0.06134) (d, f), pro-Caspase-1 (47.16% ± 0.06827) (e, i), Caspase-1 (78.19% ± 0.0134) (e, j), IL-1 β (57.09% ± 0.03169) (e, k), and P2X7R (73.04% ± 0.006164) (e, h) in C57BL/6 mouse retina were inhibited by A740003 significantly. A740003 treatment also decreased the phosphorylation of IKB α (72.25% ± 0.04076) (d, g) in C57BL/6 mouse retina. Symbols: +: with; −: without. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction ( n = 4-6).

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Activation Assay, Phospho-proteomics, Over Expression, Injection, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 inhibited the upregulation of VEGF and HIF-1 α in C57BL/6 mouse retinas injected with ox-LDL. The mRNA levels of VEGF and HIF-1 α were detected by qPCR. Compared to the vehicle-treated group, intraperitoneal injection of A740003 significantly inhibited the mRNA levels of VEGF (38.07% ± 0.01256) (a) and HIF-1 α (52.81% ± 0.01449) (b) in C57BL/6 mouse retina. Western blot showed that protein levels of VEGF (61.99% ± 0.01765) (c, d) and HIF-1 α (59.28% ± 0.02902) (c, e) in C57BL/6 mouse retina were suppressed by A740003 significantly. Symbols: +: with; −: without. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01) was determined by using one-way ANOVA with Bonferroni correction ( n = 4-6).

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Injection, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: Protective effects of A740003 on the retinal angiogenesis of C57BL/6 mice induced by ox-LDL. The retinal angiogenesis in C57BL/6 mice was assessed by immunofluorescent staining of paraffin sections of whole eyes (a). Nuclei of cells in INL and ONL were stained by DAPI (blue; (a)) and VEGF was indicated by secondary antibody (red; (a)). The average optical density of VEGF (b) was summarized. Compared to the vehicle-treated group, A740003 decreased the VEGF expression significantly in the mouse retina induced by ox-LDL. Scale bar: 50 μ m. Abbreviations: INL: inner nuclear layer; ONL: outer nuclear layer. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction ( n = 4-6).

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: A740003 decreased the retinal new blood vessels of C57BL/6 mice induced by ox-LDL. The retinal angiogenesis in C57BL/6 mice was assessed by retinal whole flat mount, and vessels were stained by isolectin-B4 (red; (a)). The numbers of new blood vessels (arrows) in retinas were summarized (b). Compared to the vehicle-treated group, A740003 reduced the new blood vessels in retinas significantly. The results were mean ± SEM. Significance of difference ( ∗∗∗ p < 0.001) was determined by using one-way ANOVA with Bonferroni correction ( n = 4-6).

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein

doi: 10.1155/2021/5520644

Figure Lengend Snippet: Retinal functions of C57BL/6 mice were assessed by dark- and light-adapted ERG. A740003 prevented the malfunction in C57BL/6 mouse retinas induced by ox-LDL. ERG was performed at 14 days after subretinal injection of ox-LDL. Representative ERG responses in the control, the ox-LDL, the ox-LDL plus vehicle-treated, and the ox-LDL plus A740003-treated groups (a) were shown. The ERG amplitudes vs. flash intensity profiles for the dark-adapted a-wave (b), dark-adapted b-wave (c), and the light-adapted b-wave (d) were summarized. Intraperitoneal injection of A740003 significantly preserved the ERG amplitudes compared to the vehicle-treated group. The results were mean ± SEM. Significance of difference ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was determined by using two-way ANOVA with Bonferroni correction ( n = 6-8).

Article Snippet: A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a final DMSO concentration no more than 1%.

Techniques: Injection, Control

Journal: Journal of Cell Science

Article Title: P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2

doi: 10.1242/jcs.175968

Figure Lengend Snippet: P2X7R inhibition blocks TG2 secretion by macrophages. (A) Differentiated monocytes express TG2. THP-1 cells were differentiated for the indicated time with TPA and stimulated with LPS as indicated. Cell extracts were analyzed by western blotting for TG2 or β-tubulin as a loading control (*, non-specific reactivity). (B) TG2 export requires P2X7R activity. Differentiated THP-1 cells were pre-treated with vehicle or 5 μM P2X7R inhibitor A740003 for 10 min, then stimulated as indicated with BzATP for 10 min with or without inhibitor (inh, pulse). Cells were chased for 30 min in P2X7R agonist and antagonist-free medium. Collected medium from the pulse and chase (200 µl) were rendered cell-free by centrifugation, and analyzed for TG2 by western blotting. (C,D) P2X7R activation triggers TG2 secretion in macrophages derived from peripheral blood mononuclear cells. Macrophages were stimulated with BzATP and chased as in B, and collected medium from the of pulse and chase were analyzed for TG2 by western blotting alongside the cell lysates (C). The presence of 100 µM Ac-YVAD-CMK did not prevent externalization or cleavage of TG2, indicating a caspase-1-independent process (D).

Article Snippet: For inhibitor studies, cells were treated with 5 μM A740003, 1 μM calmidazolium chloride (Merck-Millipore), 10 μM BAPTA-AM (Merck-Millipore) or vehicle for 10 min, and then stimulated with BzATP in the presence of the respective inhibitors as indicated.

Techniques: Inhibition, Western Blot, Control, Activity Assay, Centrifugation, Activation Assay, Derivative Assay

Journal: Journal of Cell Science

Article Title: P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2

doi: 10.1242/jcs.175968

Figure Lengend Snippet: P2X7R activation mediates TG2 externalization. (A) Analysis of TG2 secretion in HEK293 P2X7R cells. TG2-transfected cells were stimulated with BzATP or vehicle for indicated time (pulse), then incubated for 30 min in agonist-free medium (chase). TG2 secretion into cell-free supernatants was assessed by western blotting. (B) Inhibitor A740003 reversibly blocks P2X7R activation. P2X7R cells were incubated with Fluo-4-AM and 5 μM P2X7R inhibitor for 20 min prior to BzATP stimulation in the presence of inhibitor (top), washed with inhibitor-free medium for 5 min, and then re-stimulated with BzATP (bottom). The fluorescence (λ ex , 488 nm; λ em , 500–535 nm) change in individual cells was monitored by confocal microscopy (mean±s.e.m., n =30) (right). Optical sections of the same field before and 180 s after BzATP addition are shown (left). Scale bar: 25 µm. (C) P2X7R inhibitor (inh) blocks TG2 secretion. TG2-transfected P2X7R cells were pre-treated with P2X7R inhibitor or vehicle for 10 min before BzATP stimulation as indicated. TG2 release into medium was assessed as in A. (D,E) Cells release membrane-bound particles upon P2X7R activation. TG2 transfected P2X7R or parental cells were BzATP stimulated for 10 min, and chased in agonist-free medium. Conditioned media and cell lysate were analyzed by western blotting for TG2 and the microvesicle marker flotillin-2 (D) or, as a control, β-tubulin, IκBα and HMGB-1 (E).

Article Snippet: For inhibitor studies, cells were treated with 5 μM A740003, 1 μM calmidazolium chloride (Merck-Millipore), 10 μM BAPTA-AM (Merck-Millipore) or vehicle for 10 min, and then stimulated with BzATP in the presence of the respective inhibitors as indicated.

Techniques: Activation Assay, Transfection, Incubation, Western Blot, Fluorescence, Confocal Microscopy, Membrane, Marker, Control

Journal: Journal of Cell Science

Article Title: P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2

doi: 10.1242/jcs.175968

Figure Lengend Snippet: TG2 export is independent of K + efflux and membrane depolarization. (A) Calmidazolium (calm) blocks flotillin-2 but not TG2 release. TG2-transfected P2X7R cells were pre-treated for 10 min and then stimulated with BzATP in medium containing 1 µM calmidazolium or vehicle. Cells were chased in agonist-free medium, and conditioned media analyzed by western blotting for TG2 and flotillin-2. (B) Calmidazolium does not affect P2X7R-dependent ‘membrane pore’ formation. P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor A740003 or vehicle for 10 min prior to stimulation with 100 µM BzATP in the presence of respective inhibitors or carrier in PSS containing YO-PRO1 and 0.9 mM Ca 2+ . Dye uptake was monitored over time. Results are shown as mean±s.e.m. of two wells, and are representative of three independent experiments. (C) Calmidazolium ameliorates the large rise in [Ca 2+ ] i . Fluo-4-AM-loaded P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor or vehicle for 20 min prior to stimulation with 100 μM BzATP in the presence of inhibitors or carrier. Fluorescence change (λ ex , 485-12 nm; λ em , 520-10 nm) relative to control in response to agonist treatment was monitored (mean±s.e.m. of eight replicate wells).

Article Snippet: For inhibitor studies, cells were treated with 5 μM A740003, 1 μM calmidazolium chloride (Merck-Millipore), 10 μM BAPTA-AM (Merck-Millipore) or vehicle for 10 min, and then stimulated with BzATP in the presence of the respective inhibitors as indicated.

Techniques: Membrane, Transfection, Western Blot, Fluorescence, Control

Journal: Frontiers in Chemistry

Article Title: P2RX7 Purinoceptor as a Therapeutic Target—The Second Coming?

doi: 10.3389/fchem.2018.00248

Figure Lengend Snippet: Recent P2RX7 structural developments: the ATP binding pocket and the allosteric groove. (A) PyMOL (pymol.org) generated surface plot of P2RX7 trimer using RCSB PDB data file (rcsb.org) for chicken variant bound to the competitive antagonist TNP-ATP (structure 5XW6, 3.1A). ROI shows the ATP binding pocket (RHS, upper), PyMOL Video file gives structural overview of the trimer with detailed orientations of the ATP binding pocket and the central ion channel (see Supplementary Material Video ). RCSB PDB generated images show location of the ATP binding pocket in relation to the newly discovered allosteric site on the monomer (B) and at the subunit interface of the trimer, forming an allosteric groove—here shown occupied by A740003 antagonist (C) . When imaged together from intracellular projection, the three allosteric grooves appear as a Shuriken, whose rotation is impeded by the allosteric antagonist A740003 (D) . (E) Cartoon representation of the relationship between the agonist binding site and the allosteric antagonist-binding groove. Adapted from Karasawa and Kawate , with permission.

Article Snippet: This second generation antagonists began to emerge in 2006 with the development by Abbott Labs of disubstituted tetrazoles such as A438079 (Nelson et al., ) and cyanoguanidines such as A740003 (Honore et al., ).

Techniques: Binding Assay, Generated, Variant Assay