Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The sequences of oligonucleotide primers.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques:

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for immunoblotting.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for fluorescence labeling.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Fluorescence, Labeling, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

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: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

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: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

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: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

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: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

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: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

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