a740003  (Alomone Labs)

Journal: Cell Communication and Signaling : CCS

Article Title: P2X7 is an important mediator of BMP9-induced osteogenic differentiation of mesenchymal stem cells

doi: 10.1186/s12964-026-02747-w

Figure Lengend Snippet: BMP9 activates the β-catenin pathway through P2X7. A Western blot to detect the alterations in the Wnt/β-catenin signaling pathway after BMP9 and/or P2X7 overexpression treatment. B Quantitative analysis of Western blot results in Fig. 8A. C Western blot to analyze the alterations in the Wnt/β-catenin signaling pathway after BMP9 and/or P2X7 silencing treatment. D Quantitative analysis of Western blot results in Fig. 8C. E Western blot to detect the alterations in osteogenic markers, the Wnt/β-catenin signaling pathway and CaMKII phosphorylation after BMP9 treatment in the presence of A740003. F Quantitative analysis of Western blot results in Fig. 8E. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The ALP staining results were then captured using an optical microscope. (BzATP, MCE, America; A740003, Selleck, America; KN93, MCE, America).

Techniques: Western Blot, Over Expression, Phospho-proteomics

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