Journal: International Journal of Molecular Sciences

Article Title: P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β

doi: 10.3390/ijms20092298

Figure Lengend Snippet: Effects of P2X7R inhibition by 150 µM oxATP, which was added 2 h prior to 100 mU/mL BLM treatment. Equal protein amounts of cell lysates were used in SDS-PAGE and analyzed by Western blot. α-Tub served as the loading control. Untreated cells were used as the control and normalized to 100%. Representative blots from three independent experiments are shown. Charts are presented as the mean ± SEM ( n = 3) of P2X7R/ α-Tub, GSK-3β(Ser9)/ α-Tub and JAM-A/ α-Tub. P-values: P2X7R 0.0338; GSK-3β(Ser9) 0.002; and JAM-A 0.05. Immunofluorescence demonstration of JAM-A in untreated ( A ), BLM ( B ), or oxATP ( C ) treated E10 cells. Note the increased cell size after BLM exposure ( B ), which was ameliorated after oxATP ( D ). Representative images of multiple experiments ( n = 3) are shown. Bar = 20 µm. * p < 0.05, ** p < 0.01.

Article Snippet: The PVDF Immobilon-P Membrane (Merck Millipore, Billerica, USA) was blocked for 1 h in TBS-T buffer (17 mM Tris, pH 7.4; 2.7 mM KCl; 137 mM NaCl; 0.2% ( v / v ) Tween 20) including 5% ( w / v ) dried non-fat powdered milk (Carl Roth GmbH, Karlsruhe, Germany) and incubated at 4 °C overnight with the following primary antibodies: polyclonal rabbit anti-human JAM-A (A302-891A), dilution 1:1000 (Bethyl Laboratories Inc.); polyclonal rabbit anti mouse P2X7R (APR-004; Alomone Labs Inc.), dilution 1:500; monoclonal mouse anti-human GSK-3β (610202; BD Biosciences), dilution 1:1000; monoclonal rabbit anti-human Phospho-GSK-3β (Ser9) (#9323; Cell Signaling Technology Inc.), dilution 1:1000; and monoclonal mouse anti -tubulin (sc-8035; Santa Cruz Biotechnology Inc.), dilution 1:1000.

Techniques: Inhibition, SDS Page, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β

doi: 10.3390/ijms20092298

Figure Lengend Snippet: Analysis of protein levels in alveolar epithelial cell line E10 after treatment with 150 µM BzATP. Cells were treated with BzATP for 24 h and 48 h. For SDS-PAGE, equal protein amounts of cell lysates were used and analyzed by Western blot with antibodies against P2X7R, JAM-A, and α-Tub. Untreated cells were used as the control and normalized to 100%. Protein levels were normalized to α-Tub and are shown as the mean ± SEM ( n = 3) in relation to the control. One representative blot is pictured. P-values: P2X7R 0.1667 and JAM-A 0.1048.

Article Snippet: The PVDF Immobilon-P Membrane (Merck Millipore, Billerica, USA) was blocked for 1 h in TBS-T buffer (17 mM Tris, pH 7.4; 2.7 mM KCl; 137 mM NaCl; 0.2% ( v / v ) Tween 20) including 5% ( w / v ) dried non-fat powdered milk (Carl Roth GmbH, Karlsruhe, Germany) and incubated at 4 °C overnight with the following primary antibodies: polyclonal rabbit anti-human JAM-A (A302-891A), dilution 1:1000 (Bethyl Laboratories Inc.); polyclonal rabbit anti mouse P2X7R (APR-004; Alomone Labs Inc.), dilution 1:500; monoclonal mouse anti-human GSK-3β (610202; BD Biosciences), dilution 1:1000; monoclonal rabbit anti-human Phospho-GSK-3β (Ser9) (#9323; Cell Signaling Technology Inc.), dilution 1:1000; and monoclonal mouse anti -tubulin (sc-8035; Santa Cruz Biotechnology Inc.), dilution 1:1000.

Techniques: SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β

doi: 10.3390/ijms20092298

Figure Lengend Snippet: BLM treatment results in increased protein levels of P2X7R and JAM-A as well as in a reduced content of the inactive form of GSK-3β GSK-3β(Ser9). After inhibition of P2X7R by oxATP under BLM exposure, the effect on both proteins is further enhanced. Inactivating of the GSK-3β by LiCl under BLM exposure directly leads to a reduction of JAM-A. The influence of P2X7R on JAM-A is rather indirect.

Article Snippet: The PVDF Immobilon-P Membrane (Merck Millipore, Billerica, USA) was blocked for 1 h in TBS-T buffer (17 mM Tris, pH 7.4; 2.7 mM KCl; 137 mM NaCl; 0.2% ( v / v ) Tween 20) including 5% ( w / v ) dried non-fat powdered milk (Carl Roth GmbH, Karlsruhe, Germany) and incubated at 4 °C overnight with the following primary antibodies: polyclonal rabbit anti-human JAM-A (A302-891A), dilution 1:1000 (Bethyl Laboratories Inc.); polyclonal rabbit anti mouse P2X7R (APR-004; Alomone Labs Inc.), dilution 1:500; monoclonal mouse anti-human GSK-3β (610202; BD Biosciences), dilution 1:1000; monoclonal rabbit anti-human Phospho-GSK-3β (Ser9) (#9323; Cell Signaling Technology Inc.), dilution 1:1000; and monoclonal mouse anti -tubulin (sc-8035; Santa Cruz Biotechnology Inc.), dilution 1:1000.

Techniques: Inhibition

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet: P24XR and P2X7R Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

Article Snippet: Rabbit anti-mouse anti-P2X7R (extracellular) , Alomone labs , Cat# Apr-008; RRID: AB_2040065.

Techniques: Imaging, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet: Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Maximal back-projection of two representative live calcium imaging experiments performed with IFNγ- or IL4-treated RAW 264.7 cells loaded with caged-IP 3 and Fluo-4. The fluorescence variation 60 s after the irradiation of the origin cell (white box) is represented in false colors. (C) Representative traces of live calcium imaging experiments, showing the fluorescence variation after the uncaging of the origin cell (red) and the bystander macrophages (black). (D) Quantification of 3 independent live calcium imaging experiments. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

Article Snippet: Rabbit anti-mouse anti-P2X7R (extracellular) , Alomone labs , Cat# Apr-008; RRID: AB_2040065.

Techniques: Expressing, Flow Cytometry, Imaging, Fluorescence, Irradiation

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet: Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant).

Article Snippet: Rabbit anti-mouse anti-P2X7R (extracellular) , Alomone labs , Cat# Apr-008; RRID: AB_2040065.

Techniques: Incubation, Flow Cytometry, Fluorescence, Phagocytosis Assay, Derivative Assay

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse anti-P2X7R (extracellular) , Alomone labs , Cat# Apr-008; RRID: AB_2040065.

Techniques: Recombinant, Negative Control, Real-time Polymerase Chain Reaction, Software

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse anti-P2X7R (extracellular) , Alomone labs , Cat# Apr-008; RRID: AB_2040065.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Immunoprecipitation, Expressing, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Protease Inhibitor

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Mutagenesis, Transplantation Assay, Activity Assay