primary antibody for p2x4  (Alomone Labs)

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: Differential roles of P2X4 and P2X7 receptors in inducing sEV production under hypoxic conditions. CTL, CR4 (knock-down for the expression of P2X4) and CR7 (knock-down for the expression of P2X7) cells were grown under hypoxic conditions (1% O2) for a duration of 48 h prior to conducting analyses. a Size distribution and concentration of EVs purified from CTL, CR4 or CR7 cells grown under hypoxia, assessed by NTA (n = 12 independent experiments for CTL and CR4, n = 6 independent experiments for CR7). b Relative concentrations of sEVs per mL of supernatants, expressed relatively to that for CTL, in the same conditions than in a. The concentration of sEV produced by CR4 cells was significantly higher as compared to CTL (**, p <0.01). NS stands for not statistically different. c Protein concentrations of sEV extracts from same conditions than in a. There was a significantly higher protein concentration of sEVs extracts from CR4 cells, as compared to CTL (*, p <0.05) or to CR7 (*, p <0.05) cells. NS stands for not statistically different. d Relative concentration of sEVs per mL of supernatants coming from CTL cells treated with either 5-BDBD (P2X4 antagonist, 5µM) or A438079 (P2X7 antagonist, 10 µM) and expressed relatively to the CTL condition (vehicle). There was a significantly higher concentration of sEV produced by cells submitted to the P2X4 antagonist as compared to CTL (*, p <0.05) but not with cells treated with the P2X7 antagonist. NS stands for not statistically different. e Mean size (in nm) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in same conditions than in a. EVs produced by CR4 cells were significantly smaller than CTL (*, p <0.05). NS stands for not statistically different. f Zeta potential (in mV) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in similar conditions than in a. There was no difference between the three groups (n = 4 independent experiments). g Proportion of CD9+, CD63+ and CD81+ sEV purified from CR4 cells supernatants, assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 12 independent experiments). There was a significantly higher proportion of CD9+ sEV produced by CR4 cells compared to CTL cells (*, p <0.05). h Proportion of CD9+, CD63+ and CD81+ sEVs purified from supernatants of CTL cells treated with 5-BDBD (P2X4 antagonist, 5 µM), assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 8 independent experiments). There was a significantly higher proportion of CD9+ sEVs produced by cells treated with 5-BDBD (*, p <0.05). i Proportion of CD9+, CD63+ and CD81+ sEVs purified from CR7 cells, assessed by Fluo-NTA, normalized to that from CTL cells (n = 5 independent experiments). There was no statistical difference. j Proportion of CD9+-, CD63+- and CD81+-sEVs purified from CTL cells treated with A438079 (P2X7 antagonist, 10 µM), assessed by Fluo-NTA, normalized to that from CTL cells (n = 4 independent experiments). There was no statistical difference. k Intracellular expression of tetraspanins CD9, CD63, CD81 in CTL, CR4 and CR7 cells analysed by flow cytometry. Data are represented in relative MFI with mean ± SD normalized to that in 4T1 cells in CTL condition (n = 8). (**, p < 0.01). NS stands for no statistical difference. l Representative western blot showing the protein content and sEV production in CTL, CR4 and CR7 cells, under both normoxia and hypoxia. Markers of sEVs (CD9, CD63, CD81, TSG101, Flotillin1, Syntinin-1), are enriched in extracellular vesicle fractions, but not GRP94 used as a marker of endoplasmic reticulum which was only identified in total cell protein extract. sEVs produced by CR4 cells demonstrated a higher level of CD9. HSC70 and β-actin were used as control proteins

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Knockdown, Expressing, Concentration Assay, Purification, Produced, Protein Concentration, Zeta Potential Analyzer, Flow Cytometry, Western Blot, Marker, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: EVs from P2X4-deficient cells show a differential expression of proteins involved in endo-lysosome, exosome and autophagosome fusion and maturation. a Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins implicated in late endosome/MVBs transport and fusion to lysosome (GO term reported Supplementary Fig 5b), identified in EVs coming from CTL and CR4 (n=3 independent experiments). *, p < 0.5; **, p < 0.01; *** p < 0.001. b STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CTL cells, thus expressing P2X4, and involved in late endosome/MVBs transport or fusion to lysosome. c Heatmap illustrating the differential expression (z-score, differences in Log2-fold change and p value) of proteins involved in early endosome transport, endosome maturation, autophagy and exosome assembly (GO term reported Supplementary Fig 5a), identified in EVs coming from CTL and CR4 (n=3 independent experiments). Statistically different at: *, p < 0.5; **, p < 0.01 and ***, p < 0.001. d STRING analysis showing the interaction of selected proteins identified as being upregulated in EVs from CR4 cells, thus not expressing P2X4, involved in exosome assembly, autophagy and early endosome transport, endosome maturation. e Representative western blot showing the presence of autophagy markers LC3-II and P62 in CTL, CR4 and CR7 cells, cultivated either under normoxia or hypoxia, and their respective purified EVs

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: sEVs produced from P2X4-expressing cancer cells enhance the invasive capacities of recipient cancer cells. a CTL cancer cells were treated with EVs purified from CTL, CR4 or CR7 cells, grown under hypoxic conditions (1% O2). EVs were stained with green, fluorescent membrane probe PKH67. Representative fluorescent images obtained after 4 h treatment with 20 µg/mL of EVs. Upper, merged images showing DAPI staining for identification of cell nuclei and PKH67 staining showing EVs. Lower, PKH67 staining alone. White arrows indicate EVs incorporated into CTL recipient cells. Scale bars, 75 μm. b Assessment of the uptake as a function of time of PKH67-stained EVs, produced by CTL, CR4, CR7 cells, in CTL cancer recipient cells by flow cytometry (treatment with 20 µg/mL EVs protein). Results indicate a speed of uptake expressed as a ratio of the uptake after 4-h on that after 1-h incubation. Data are represented in rMFI with Mean MFI 4 h normalized to their respective control MFI 1 h, mean ± SD (n = 6 independent experiments). *, indicate a statistical difference at p < 0.05. NS stands for no statistical difference. c Cartoon of the experimental procedure of co-culture assays to assess cell invasiveness performed in hypoxic conditions (1% O2). Invasiveness of recipient CTL was assessed as being their capacity to invade an 8 μm-sized filter covered by a thin layer of Matrigel. These cells were co-cultured with CTL, CR4, CR7 donor cells seeded at the bottom of the well. d Invasive capacities of recipient CTL cells co-cultured with CTL, CR4 or CR7 donor cells, as illustrated in c. Experiments were performed in the absence (vehicle, DMSO) or presence of the nSMAse inhibitor GW4869 (10 µM). Data are normalized to the control condition, in absence of GW4869, and presented as mean ± SD ( n = 6 independent experiments). *, p < 0.05 and **, p < 0.01. NS stands for no statistical difference

Article Snippet: Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X-100 for 15 min. Unspecific antibody binding was blocked with 3% BSA for 20 min. Primary antibody for P2X4 (Alomone, APR-002, 1:200) was incubated for 2 h at room temperature.

Techniques: Produced, Expressing, Purification, Staining, Membrane, Flow Cytometry, Incubation, Control, Co-Culture Assay, Cell Culture

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