anti p2x4 antibody  (Alomone Labs)

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effect of P2X4 receptors in TAMs on the cGAS-STING pathway and mtDNA release (A) PBMC-derived macrophages were induced into TAMs by SW480-conditioned medium with the addition of 20 μmol/L BAY-1797 (P2X4 blocker), 20 μmol/L A-740003 (P2X7 blocker), and 20 μmol/L NF279 (P2X1 blocker), respectively. An equal final concentration of DMSO (0.05%) was used including vehicle controls. The STAT1 phosphorylation were observed by western blot. (B) P2X4 receptor expression in M1/M2 macrophages was observed by cytometry. Median fluorescence intensity (MFI) was compared (∗∗ p < 0.01, One-way ANOVA). (C) Lentiviral transfection was performed to construct P2X4 overexpression or knockdown stable cell lines of THP-1 cells, the expression of P2X4 protein was verified. The differences in protein phosphorylation were compared among TAMs derived from each stable cell line by western blotting. (D) The surface markers of macrophage (CD80-APC, CD163-PE) were compared by cytometry (∗∗ p < 0.01, One-way ANOVA). (E) The mRNA levels of M1 marker genes and cytokine secretion levels were compared via qPCR and ELISA (∗∗ p < 0.01, Kruskal-Wallis test). (F) The mRNA levels of M2 marker genes were compared via qPCR (Kruskal-Wallis test). All data are presented as mean ± SD. Data points represent independent biological replicates. Western blots images shown are representative of 3 independent experiments.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Derivative Assay, Concentration Assay, Phospho-proteomics, Western Blot, Expressing, Cytometry, Fluorescence, Transfection, Construct, Over Expression, Knockdown, Stable Transfection, Marker, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Mechanism of mitochondrial calcium overload by P2X4 receptor-mediated calcium influx in TAMs (A) TAMs were induced from THP-1-derived macrophages. Cytosolic dsDNA was detected using an anti-dsDNA antibody with Alexa Fluor 488 (green), and nuclei were counterstained with DAPI (blue). Images were acquired by confocal microscopy (scale bars, 10 μm). Representative image shown ( N = 3 independent experiments, 10 images were analyzed per experiment). The boxplot shows the signal intensity (∗∗ p < 0.01, Student’s t test). (B) BAY-1797 was added during induction of TAMs. The trends of mtDNA/nDNA changes in the cytoplasm of cells were compared by detecting reference genes via qPCR (∗∗ p < 0.01, Student’s t test). (C) After incubation THP-1-derived macrophages from P2X4-OE, shP2X4, and control groups with the Ca 2+ probe Rhod-2, the magnitude of Ca 2+ concentration fluctuations in response to 100 μM eATP stimulation was measured on a fluorometric plate reader. (D) The THP-1 stable cell lines were induced into TAMs and Ca 2+ fluorescence (scale bars, 10 μm) was observed under a confocal microscope ( N = 3 independent experiments). (E) THP-1-derived macrophages (5 biological samples per group) were loaded with Rhod-2 probe, mitoSOX probe or JC-1 probe, respectively. After 3 h of induction by adding SW480-conditioned medium, intracellular Ca 2+ concentration, mitochondrial ROS levels and mitochondrial membrane potential changes were detected. All experiments were independently performed three times. (F) During induction of TAM, simultaneous NAC intervention was set up or calcium-free SW480-conditioned medium was used. Intracellular Ca 2+ concentration and mitochondrial ROS levels were detected. Differences in the mtDNA/nDNA ratio in the cytoplasm were compared (∗ p < 0.05, ∗∗ p < 0.01, One-way ANOVA). (G) After TAM induction from THP-1-derived macrophages stably overexpressing FLAG-cGAS, cGAS ChIP was performed and mtDNA/nDNA enrichment was compared by detecting reference genes via qPCR (∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Derivative Assay, Confocal Microscopy, Incubation, Control, Concentration Assay, Stable Transfection, Fluorescence, Microscopy, Membrane

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effects of P2X4 knockout on the polarization of TAMs and T cell regulation (A) P2X4 knockout Ana-1 cell lines were constructed. western blot detected P2X4 expression in three of the monoclonal cell lines. Western blots shown are representative of 3 independent experiments. (B) Wild type or P2X4-K.O. Ana-1 cells were induced into TAMs by MC38 conditioned medium. The mRNA expression of M1/M2 cytokines were compared by qPCR 48 h later (∗ p < 0.05, ∗∗ p < 0.01, One-way ANOVA). (C) Suspension mononuclear cells isolated from the spleen of C57BL/6J mice were grouped to be co-cultured with TAMs (sgP2X4) or TAMs (WT). Living cells were counted using a cell viability analyzer at time points (∗∗ p < 0.01, Student’s t test). (D) After 72 h, the suspension cells were harvested and stained with CD3 (PE-Cy7), CD4 (PE), CD8 (FITC), and CXCR6 (APC) for flow cytometry. Another set of cells were fixed for GzmB (BV421) staining after stimulated with a combination of 50 ng/mL PMA, 1 μg/mL ionomycin, and 5 μg/mL brefeldin A for 4 h. Data were compared by histogram (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Knock-Out, Construct, Western Blot, Expressing, Suspension, Isolation, Cell Culture, Staining, Flow Cytometry

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effect of P2X4 knockout macrophages on T cell infiltration in MC38 tumors in mice (A) 6-week C57BL/6J female mice were divided into 3 groups and inoculated as described. Tumor volume and weight were compared (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). (B) Subcutaneous tumors in each group (4 per group, randomly selected) were digested into single-cell suspensions and stained with antibodies against CD45 (PE-Cy7), F4/80 (AF647), CD80 (PE-CF594), and CD86 (PE) for macrophages, or with antibodies against CD3 (PE-Cy7), CD4 (PE), CD8 (FITC), CXCR6 (APC) and GzmB (BV421) for T cells before flow cytometry. (C) The total number of tumor infiltrating immune cells was measured and the proportions of TAMs and T cells in each group were compared by histogram. (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Knock-Out, Staining, Flow Cytometry

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Characterization of P2X4 receptor expression and prognosis in CRC (A) P2X4 protein expression in tumor tissue (T) and adjacent normal tissue (N) from 24 CRC patients was detected by western blot. Relative P2X4 expression were quantified by densitometry and compared in a paired dot plot (∗∗ p < 0.01, paired Student’s t test). (B) Immunofluorescence staining (P2X4-AF488, CD68-AF594) was performed and observed by confocal microscopy (scale bars, 50 μm). Representative images shown from paired CRC and adjacent tissues ( n = 12 patients). Boxplot below indicates the number of P2X4 + CD68 + cells per field of view (∗∗ p < 0.01, Student’s t test). (C) 457 COAD patients in the TCGA database were divided into P2X4 high and low expression groups using FPKM = 3.67 as a cut-off, and overall survival was compared ( p = 0.014, Log rank test). (D) Correlation between P2X4 and CXCR6 expression in 623 CRC patients from TCGA database (R = 0.31, p < 0.01, Spearman Analysis). Data are presented as mean ± SD. Each dot represents one individual patient sample.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Confocal Microscopy