Journal: Purinergic Signalling

Article Title: P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

doi: 10.1007/s11302-017-9567-2

Figure Lengend Snippet: P2X7, but not P2X4 receptor, mediates ASN-induced calcium influx. a SH-SY5Y cells were preincubated for 60 s with 10 μM AZ 11645373 (P2X7 receptors antagonist) or 100 μM 5-BDBD (P2X4 receptor antagonist) in HBSS. Baseline Fluo-4 fluorescence was monitored for 60 s prior to the addition of ASN (10 μM). Fluorescence was monitored for a further 300 s, and values were converted to %F/F0, where F0 is the fluorescence value of first record (0 s). b Responses were quantitated by measuring the AUC value. The results are presented as the mean ± SEM from three to six independent experiments ( n = 3–6). ### p < 0.001 versus ASN-treated cells using a one-way ANOVA followed by the Bonferroni test. c The SH-SY5Y cells were preincubated with 10 μM AZ 11645373 or 100 μM PPADS for 5 min in the Locke 5 medium containing 5 mM HEPES (pH 7.4), followed by a 5 min incubation with 10 μM ASN or 5 mM ATP. Then, [45] CaCl 2 (1 μCi/well) was added for 10 min. The results are presented as the mean ± SEM from three to six independent experiments ( n = 3–6). *** p < 0.001 versus control, ### p < 0.001 versus ASN-treated cells, &&& p < 0.001 versus ATP-treated cells using a one-way ANOVA followed by the Bonferroni test. d Rat brain synaptoneurosomes were preincubated with PPADS (100 μM) or AZ 11645373 (10 μM) for 5 min, followed by a 30-min incubation with 10 μM ASN or 5 mM ATP at 37 °C in HBSS medium (pH 7.4). The reaction was started by adding 0.1 μCi [45] CaCl 2 . The results are presented as the mean ± SEM from three to six independent experiments ( n = 3–6). ** p < 0.01 versus control, # p < 0.05 versus ASN-treated cells, &&& p < 0.001 versus ATP-treated cells using a one-way ANOVA followed by the Bonferroni test

Article Snippet: Further, membranes were washed three times for 5 min in TBST and incubated with the following primary antibody: rabbit monoclonal anti-P2X7R (Sigma-Aldrich, cat. P8232; 1∶200) [ ] in a 5% BSA solution in TBST, overnight at 4 °C and rabbit monoclonal anti-P2Y1 (Sigma-Aldrich, cat. P6487; 1∶200) [ ] in TBST overnight at 4 °C.

Techniques: Fluorescence, Incubation

Journal: Purinergic Signalling

Article Title: P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

doi: 10.1007/s11302-017-9567-2

Figure Lengend Snippet: Activation of P2X7 receptors leads to pannexin 1-dependent ATP release. a Extracellular ATP level was analyzed after 1-min treatment with 10 μM ASN together or without 10 μM CBX (antagonist of pannexin channels) or 10 μM AZ 11645373 (P2X7 receptors antagonist) at 37 °C in HBSS with 5 mM HEPES (pH 7.4) using the luciferase-based protocol described in the “ ” section. Data, calculated from respective calibration curves, are expressed as nM ATP and the data represent the mean value ± SEM for six to eight separate experiments ( n = 6–8). *** p < 0.001 versus control, ### p < 0.001 versus ASN-treated cells using a one-way ANOVA followed by the Bonferroni test. b SH-SY5Y cells were preincubated (60 s) with 10 μM CBX or appropriate solvent in HBSS. Baseline Fluo-4 fluorescence was monitored for 60 s prior to the addition of ASN (10 μM) or appropriate solvent. Fluorescence was monitored for a further 300 s, and values were converted to %F/F0, where F0 is the fluorescence value of first record (0 s). c Responses were quantitated by measuring the AUC value. The results are presented as the mean ± SEM from four to six independent experiments ( n = 4–6). *** p < 0.001 versus control using a one-way ANOVA followed by the Bonferroni test. d Rat brain synaptoneurosomes were preincubated with CBX (10 μM) for 5 min, followed by a 30-min incubation with 10 μM ASN at 37 °C in HBSS medium (pH 7.4). The reaction was started by adding 0.1 μCi [45] CaCl 2 . The results are presented as the mean ± SEM from three to six independent experiments ( n = 3–6). ** p < 0.01, *** p < 0.001 versus control using a one-way ANOVA followed by the Bonferroni test

Article Snippet: Further, membranes were washed three times for 5 min in TBST and incubated with the following primary antibody: rabbit monoclonal anti-P2X7R (Sigma-Aldrich, cat. P8232; 1∶200) [ ] in a 5% BSA solution in TBST, overnight at 4 °C and rabbit monoclonal anti-P2Y1 (Sigma-Aldrich, cat. P6487; 1∶200) [ ] in TBST overnight at 4 °C.

Techniques: Activation Assay, Luciferase, Fluorescence, Incubation

Journal: Purinergic Signalling

Article Title: P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

doi: 10.1007/s11302-017-9567-2

Figure Lengend Snippet: The increase in extracellular ATP by ASN is not responsible for P2X7 receptor activation. a Extracellular ATP level was analyzed after 1-min treatment with 10 μM ASN together or without 0.5, 1, or 5 U apyrase at 37 °C in HBSS with 5 mM HEPES (pH 7.4) using the luciferase-based protocol described in the “ ” section. Data, calculated from respective calibration curves, are expressed as nM ATP and the data represent the mean value ± SEM for three separate experiments ( n = 3). *** p < 0.001 versus control, ### p < 0.001 versus ASN-treated cells using a one-way ANOVA followed by the Bonferroni test. b SH-SY5Y cells were preincubated (60 s) with 1 U apyrase with or without 10 μM AZ 11645373 (P2X7 receptors antagonist) or appropriate solvent in HBSS. Baseline Fluo-4 fluorescence was monitored for 60 s prior to the addition of ASN (10 μM) or appropriate solvent. Fluorescence was monitored for a further 300 s, and values were converted to %F/F0, where F0 is the fluorescence value of first record (0 s). c Responses were quantitated by measuring the AUC value. The results are presented as the mean ± SEM from four independent experiments ( n = 4). *** p < 0.001 versus control, ### p < 0.001 versus ASN-treated cells using a one-way ANOVA followed by the Bonferroni test. d Rat brain synaptoneurosomes were preincubated with 1 U apyrase with or without 10 μM AZ 11645373 for 5 min, followed by a 30-min incubation with 10 μM ASN at 37 °C in HBSS medium (pH 7.4). The reaction was started by adding 0.1 μCi [45] CaCl 2 . The results are presented as the mean ± SEM from three independent experiments ( n = 3). *** p < 0.001 versus control, ### p < 0.001 versus ASN-treated cells using a one-way ANOVA followed by the Bonferroni test

Article Snippet: Further, membranes were washed three times for 5 min in TBST and incubated with the following primary antibody: rabbit monoclonal anti-P2X7R (Sigma-Aldrich, cat. P8232; 1∶200) [ ] in a 5% BSA solution in TBST, overnight at 4 °C and rabbit monoclonal anti-P2Y1 (Sigma-Aldrich, cat. P6487; 1∶200) [ ] in TBST overnight at 4 °C.

Techniques: Activation Assay, Luciferase, Fluorescence, Incubation

Journal: Molecular Cancer

Article Title: The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells

doi: 10.1186/s12943-015-0472-4

Figure Lengend Snippet: Expression of P2X7R in PDACs and HPDE cells. a . Real time PCR and RT-PCR analysis of P2X7R expression in HPDE and PDAC cells. Insert shows a representative gel of P2X7R mRNA (284 bp) in Panc-1. The data were normalized with respect to the three housekeeping genes: β-actin , β glucuronidase ( GUSB ) and glutaminyl-tRNA synthetase ( QAR S) and expression in HPDE was set to 1. The bargraph shows data for four experiments (mean ± SEM). The relative amount of mRNA was calculated from a standard curve run on each plate. b . Western blot band at around 70 kDa corresponding to the isoform A [PR: Q99572-1], as shown on the insert. Loading control was β-actin [PR: P60709] detected at 42 kDa. All lanes were loaded with 50 μg of proteins. c . Western blot band at around 42 kDa most likely corresponds to the isoform B [PR: Q99572-2]. The results were normalized with β-actin that was used as a loading control. The graphs for the two isoforms show data of three experiments (mean ± SEM). Significant differences of expression in PDAC in comparison to HPDE cells P < 0.05 (*) and P < 0.001 (**) are indicated

Article Snippet: Cells were incubated with antibody against P2X7R extracellular loop (1:100 rabbit polyclonal, Alomone, APR-008) overnight at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Comparison

Journal: Molecular Cancer

Article Title: The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells

doi: 10.1186/s12943-015-0472-4

Figure Lengend Snippet: Immunolocalization of P2X7R in PDACs and HPDE cells. HPDE and Panc-1 cells were grown on coverslips. P2X7R was stained with a polyclonal antibody against the extracellular domain and Alexa Fluor 568 (red) and DAPI was used to stain the nucleus (blue). All bars are 25 μm

Article Snippet: Cells were incubated with antibody against P2X7R extracellular loop (1:100 rabbit polyclonal, Alomone, APR-008) overnight at 4 °C.

Techniques: Staining

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: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Oligonucleotide Primer List.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Antibody List.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Binding Assay, Transduction

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: To assess whether P2X7-R splice variants other than the full length, variant 1 are expressed in the WT and P2X7-KO mouse retina and also whether read through of variant 1 C-terminal mRNA occurs in the P2X7-KO mouse retina, PCR for (A) the central region of the P2X7-R which is common to variants 1, 2 and 3; (B) the C-terminus from within the start of the disruption in the P2X7-KO to the end of the mRNA (variant 1 only, primer pair A); and (C) the C-terminus from after the insertion deletion in the P2X7-KO mouse to the end of the receptor (variant 1 only, primer pair B) was completed. P2X7-R variant 1 C-terminal mRNA was absent (B–C) in the P2X7-KO mouse but N-terminal mRNA variants (likely variant 2 or 3) were still expressed (A). MW, molecular weight marker and the bright bar is 300 bp. (D–E) Protein analysis of P2X7-R splice variant expression was completed using Western blots of mouse retina (D) and cortex (E). An N-terminal specific P2X7-R antibody detected the protein transcript of variant 1 mRNA in WT samples 1 and 2 but not P2X7-KO samples 3 and 4 (68.41 kDa) of both retina and cortex. Protein for variant 2/3 (50.69 kDa/49.38 kDa) and 4 (17.2 kDa) was expressed in both WT and P2X7-KO retina and cortex. A monoclonal antibody against GAPDH was applied to the same blots and indicates that similar amounts of protein were loaded for WT and P2X7-KO samples.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Variant Assay, Molecular Weight, Marker, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Mice were dark adapted overnight and mixed (rod and cone) ERG responses were recorded in response to full field flashes from intensity −4.2 to 2.1 log cd.s/m 2 . (A–B) Representative mixed ERG waveforms for (A) WT and (B) P2X7-KO mice are presented. (C) There was no change in the amplitude of the rod photoreceptor derived component, a-wave, as a function of intensity. (D) The amplitude of the post-photoreceptor response, the b-wave was larger in P2X7-KO mice than in WT mice across almost all intensities tested. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the rod ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The photoreceptor derived, rod PIII (modelled a-wave) was not altered in either (B) amplitude or (C) sensitivity in the P2X7-KO mouse when compared with the WT response. The post-photoreceptor, rod PII (modelled b-wave) was found to be (D) larger in amplitude and (E) faster to reach a maximum response. (F) The amplitude of the third of the inner retinal derived oscillatory potentials was significantly enhanced in the P2X7-KO mouse. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the cone ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The post-photoreceptor, cone PII (modelled b-wave) was found to be larger in (B) amplitude but not faster to reach a maximum response (C) in the P2X7-KO mouse when compared with the WT response. (D) The photopic visual acuity, assessed using the optokinetic reflex to determine the spatial frequency threshold, was higher in the P2X7-KO mouse than in the WT. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Transverse sections of paraffin embedded retina from (A) WT and (B) P2X7-KO mice were stained with hematoxylin and eosin for comparison. The gross retinal morphology was similar in both mice. Scale bar, 20 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Staining

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Immunohistochemistry on a transverse retinal section from a WT mouse showing P2X7-R immunoreactivity in the outer plexiform layer (OPL), inner nuclear layer (INL), and ganglion cell layer (GCL). (C) The retina does not label with the P2X7-R antibody following incubation with P2X7-R specific peptide. Scale bar, 20 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Immunohistochemistry, Incubation

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (green) colocalises with horizontal cells labelled with Calbindin (red) and their terminals. (B) P2X7-R IR (green) colocalises with rod bipolar cells labelled with PKC (red). (C) Inset of rod bipolar cell terminals. (D) P2X7-R IR (green) puncta are discretely expressed within photoreceptor terminals labelled with PSD-95 (red). (E) Cone bipolar cells (Type 2 and 6) labelled with a ZNP-1 antibody (green) colocalise with P2X7-R IR (red) including both the cone bipolar cell dendrites and terminals (F). P2X7-R IR (green) colocalises with amacrine cells labelled with calretinin (G) and in particular GABAergic amacrine cells (H). (I) Ganglion cells labelled with an antibody against GFP in the Thy1-HYFP mouse (pseudo-coloured red) were P2X7-R IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (red) is associated with but does not colocalise with ribbon synapses (ribeye, green) on bipolar cell terminals (VGLUT, blue). (B) Magnified inset from A, arrows indicate ribbon synapses associated with P2X7-R IR puncta. (C) P2X7-R IR (red) colocalises with conventional synapses (bassoon, green) distinct from bipolar cell terminals (VGLUT, blue). (D) Magnified inset from C, arrows indicate conventional synapses that colocalise with P2X7-R IR puncta. Co-localisation analysis indicates that P2X7-R IR puncta do not co-localise with ribbon synapses (E) but do show significant colocalisation with conventional synapses (F) in the IPL. Scale bars are 5 microns for A & C and 2 microns for B & D.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Ribbons are indicated by arrows and labelled amacine cells are indicated by astericks. (A), (B) & (C) P2X7-R immunoreactivity labels amacrine cells adjacent to rod bipolar cell ribbon synapses in the IPL. (D) A conventional amacrine cell synapse is labelled for P2X7 immunoreactivity in close proximity to a rod bipolar cell terminal. Scale bars are 200 nm for A & B and 500 nm for C & D.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A–B) Müller cells, labelled with an antibody against glutamine synthetise (red) were found to be closely associated with P2X7-R-IR puncta in the IPL (green), but not co-localised in the IPL. (C) Astrocytes, labelled with glial fibrilliary acidic protein (GFAP; red) and P2X7-R-IR (green) were found to co-localise in retinal flat mount. (D) Microglia labelled with mouse anti-GFP (pseudo-coloured red) from CX3CR1-GFP heterozygous mice were also found to co-localise with P2X7-R-IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: Frontiers in Medicine

Article Title: The Role of the Superior Cervical Sympathetic Ganglion in Ischemia Reperfusion-Induced Acute Kidney Injury in Rats

doi: 10.3389/fmed.2022.792000

Figure Lengend Snippet: Primers used for real-time PCR.

Article Snippet: The membranes were blocked in 5% skim milk in TBST for 2 h at room temperature and then incubated with primary antibody overnight at 4 ° C. Primary antibodies were applied as follows: rabbit anti-TNF-α (1:1000; Cat No. 17590-1-AP; Proteintech Group Co., Ltd., China), rabbit anti-IL-6 (1:1000; Cat No. A0286; Abclonal Technology Co., Ltd., China), rabbit anti-TH (1:1,000; #58844S; Cell Signaling Technology, United States), rabbit anti-Bcl2 (1:1,000; Cat No. A11313; Abclonal Technology Co., Ltd., China), rabbit anti-BAX (1:1,000; Cat No. A0207; Abclonal Technology Co., Ltd., China), rabbit anti-Caspase-3 (1:1,000; Cat No. A19664; Abclonal Technology Co., Ltd., China), rabbit anti-P2X3R (1:1,000; Cat No. A12965; Abclonal Technology Co., Ltd., China), rabbit anti-P2X7R (1:1,000; Cat No. A10511; Abclonal Technology Co., Ltd., China), rabbit anti-GAPDH (1:10,000; Cat No. BM1623; Wuhan Boster Biological Technology, Ltd., China), rabbit anti-β-actin (1:100,000; Cat No. AC026; Abclonal Technology Co., Ltd., China).

Techniques:

Journal: Frontiers in Medicine

Article Title: The Role of the Superior Cervical Sympathetic Ganglion in Ischemia Reperfusion-Induced Acute Kidney Injury in Rats

doi: 10.3389/fmed.2022.792000

Figure Lengend Snippet: The activated purinergic P2X receptor in the spinal cord upon sympathetic denervation. (A,B) Real-Time PCR for P2X3R and P2X7R in the spinal cord. β-actin used as internal control. (C) Representative images of protein levels of P2X3R and P2X7R. (D,E) The expression levels of P2X3R and P2X7R in the spinal cord were normalized to GAPDH within the same sample. * P < 0.05; ** P < 0.01, each group contains at least 4 rats.

Article Snippet: The membranes were blocked in 5% skim milk in TBST for 2 h at room temperature and then incubated with primary antibody overnight at 4 ° C. Primary antibodies were applied as follows: rabbit anti-TNF-α (1:1000; Cat No. 17590-1-AP; Proteintech Group Co., Ltd., China), rabbit anti-IL-6 (1:1000; Cat No. A0286; Abclonal Technology Co., Ltd., China), rabbit anti-TH (1:1,000; #58844S; Cell Signaling Technology, United States), rabbit anti-Bcl2 (1:1,000; Cat No. A11313; Abclonal Technology Co., Ltd., China), rabbit anti-BAX (1:1,000; Cat No. A0207; Abclonal Technology Co., Ltd., China), rabbit anti-Caspase-3 (1:1,000; Cat No. A19664; Abclonal Technology Co., Ltd., China), rabbit anti-P2X3R (1:1,000; Cat No. A12965; Abclonal Technology Co., Ltd., China), rabbit anti-P2X7R (1:1,000; Cat No. A10511; Abclonal Technology Co., Ltd., China), rabbit anti-GAPDH (1:10,000; Cat No. BM1623; Wuhan Boster Biological Technology, Ltd., China), rabbit anti-β-actin (1:100,000; Cat No. AC026; Abclonal Technology Co., Ltd., China).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Molecular Medicine Reports

Article Title: Role of P2X7R in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps

doi: 10.3892/mmr.2021.12160

Figure Lengend Snippet: Haematoxylin and eosin staining of nasal mucosa and immunolabelling of P2X7R. Numerous eosinophils infiltrated the nasal mucosa of ECRSwNP. (A) Immunofluorescence staining showed that P2X7R was predominantly expressed in epithelial cells and P2X7R protein levels were significantly overexpressed in CRSwNP groups, especially in the ECRSwNP group vs. control group. (B and C) Data represent means ± SD. **P<0.01 and ***P<0.001. CRSwNP, chronic rhinosinusitis with nasal polyps; ECRSwNP, eosinophilic CRSwNP; P2X7R, purinergic 2X7 receptor.

Article Snippet: The membranes were blocked with 5% non-fat milk at 4°C for 1 h and then incubated with the working dilution of primary antibodies: Rabbit anti-human P2X7R (1:1,000; GeneTex; cat no. GTX16827); rabbit anti-human NLRP3 (1:1,000; Abcam; cat no. ab260017); mouse anti-human IL-1β (1:5,000; Arigo Biolaboratories; cat. no. ARG66285); rabbit anti-human GAPDH (1:5,000; AntGene; cat. no. ANT012) at 4°C overnight.

Techniques: Staining, Immunofluorescence, Control

Journal: Molecular Medicine Reports

Article Title: Role of P2X7R in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps

doi: 10.3892/mmr.2021.12160

Figure Lengend Snippet: Expression profile of P2X7R in nasal mucosa. (A) Western blotting analysis showed that P2X7R, (B) NLRP3 (C) and IL-1β (D) protein levels were significantly increased in the CRSwNP groups compared with the control group, and were significantly more highly expressed in the ECRSwNP group compared with the NECRSwNP group. The mRNA expression levels of P2X7R and IL-1β were elevated in the CRSwNP groups compared with the control group, and these increases were also found in the ECRSwNP group compared with the NECRSwNP group. (E and F) *P<0.05; **P<0.01; ***P<0.001. CRSwNP, chronic rhinosinusitis with nasal polyps; NECRSwNP, non-eosinophilic CRSwNP; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; P2X7R, purinergic 2X7 receptor; NLRP3, NLR family pyrin domain-containing 3; IL-1β, interleukin-1β.

Article Snippet: The membranes were blocked with 5% non-fat milk at 4°C for 1 h and then incubated with the working dilution of primary antibodies: Rabbit anti-human P2X7R (1:1,000; GeneTex; cat no. GTX16827); rabbit anti-human NLRP3 (1:1,000; Abcam; cat no. ab260017); mouse anti-human IL-1β (1:5,000; Arigo Biolaboratories; cat. no. ARG66285); rabbit anti-human GAPDH (1:5,000; AntGene; cat. no. ANT012) at 4°C overnight.

Techniques: Expressing, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Role of P2X7R in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps

doi: 10.3892/mmr.2021.12160

Figure Lengend Snippet: Inflammatory stimulation with LPS combined with BzATP was inhibited by A740003. (A) Western blot analysis of the expression levels of P2X7R (B) and NLRP3 (C) in HNEC after A740003 inhibition of inflammation induced by LPS+BzATP. The expression of P2X7R and NLRP3 was significantly increased in response to LPS plus BzATP; furthermore, the selective P2X7R antagonist, A740003, suppressed this process, decreasing the protein expression of NLRP3 and P2X7R. (D and E) The mRNA expression levels of (D) P2X7R and (E) IL-1β were decreased after stimulation with LPS+BzATP and subsequent inhibition with A740003. (F) Enzyme-linked immunosorbent assay results show the changes in the concentration of IL-1β in the cell culture supernatant. *P<0.05 and **P<0.01. NLRP3, NLR family pyrin domain-containing 3; LPS, lipopolysaccharides; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt; IL-1β, interleukin-1β; P2X7R, purinergic 2X7 receptor.

Article Snippet: The membranes were blocked with 5% non-fat milk at 4°C for 1 h and then incubated with the working dilution of primary antibodies: Rabbit anti-human P2X7R (1:1,000; GeneTex; cat no. GTX16827); rabbit anti-human NLRP3 (1:1,000; Abcam; cat no. ab260017); mouse anti-human IL-1β (1:5,000; Arigo Biolaboratories; cat. no. ARG66285); rabbit anti-human GAPDH (1:5,000; AntGene; cat. no. ANT012) at 4°C overnight.

Techniques: Western Blot, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Molecular Medicine Reports

Article Title: Role of P2X7R in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps

doi: 10.3892/mmr.2021.12160

Figure Lengend Snippet: LPS activates Toll-like receptor 4, leading to transcription of the IL-1β gene and translation of the 31-kDa pre-cytokine (pro-IL-1β). P2X7R exposure to its agonist, BzATP, triggers depolarization via the opening of cation channels, which triggers K + efflux, leading to stimulation of the NLRP3 inflammasome. Activated caspase-1 converts pro-IL-1β to the 17-kDa mature protein (IL-1β), which is then secreted to the extracellular space. A740003, a selective competitive antagonist of P2X7R, blocks P2X7R-mediated calcium influx, pore formation and IL-1β release. TLR4, Toll-like receptor 4; IL-1β, interleukin-1β; P2X7R, purinergic 2X7 receptor; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt; NLRP3, NLR family pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membranes were blocked with 5% non-fat milk at 4°C for 1 h and then incubated with the working dilution of primary antibodies: Rabbit anti-human P2X7R (1:1,000; GeneTex; cat no. GTX16827); rabbit anti-human NLRP3 (1:1,000; Abcam; cat no. ab260017); mouse anti-human IL-1β (1:5,000; Arigo Biolaboratories; cat. no. ARG66285); rabbit anti-human GAPDH (1:5,000; AntGene; cat. no. ANT012) at 4°C overnight.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

doi: 10.1186/s12974-024-03057-z

Figure Lengend Snippet: P2X7R expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01

Article Snippet: Following fixation and permeabilization, the cells were further stained with rabbit anti-human P2X7R antibody (Boster Biological Technology) or rabbit anti-human NLRP3 antibody (Thermo Fisher) for 40 min at room temperature, followed by incubation with FITC-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific) for an additional 40 min at room temperature.

Techniques: Expressing, Isolation, Flow Cytometry, Quantitative RT-PCR, Control

Journal: Journal of Neuroinflammation

Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

doi: 10.1186/s12974-024-03057-z

Figure Lengend Snippet: P2X7R expression was significantly elevated in CD4 + T cells in EAN rats. P2X7R expression in T cells and macrophages in control (unimmunized group) and EAN rats at day 18. (A-B) Fluorescence photomicrographs showing P2X7R expression and quantification in CD4 + T cells, CD8 + T cells and macrophages (CD68 + ) in the sciatic nerves of EAN and control rats. Scale bars, 20 μm. (C) Graph showing the number of P2X7R-positive cells in sciatic nerve sections obtained from control and EAN rats. (D-E) Flow cytometry analysis of P2X7R expression and quantification on CD4 + T cells, CD8 + T cells and macrophages in the spleen of EAN and control rats. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. * p < 0.05, ** p < 0.01

Article Snippet: Following fixation and permeabilization, the cells were further stained with rabbit anti-human P2X7R antibody (Boster Biological Technology) or rabbit anti-human NLRP3 antibody (Thermo Fisher) for 40 min at room temperature, followed by incubation with FITC-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific) for an additional 40 min at room temperature.

Techniques: Expressing, Control, Fluorescence, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

doi: 10.1186/s12974-024-03057-z

Figure Lengend Snippet: BBG treatment suppressed the P2X7R/NLRP3 pathway in CD4 + T cells in EAN. Proteins were obtained from sciatic nerves at the peak of EAN and subjected to western blot analysis and quantification. NLRP3 expression in immune cells was analyzed through flow cytometry and immunofluorescence. (A) Representative western blots of P2X7R, NLRP3, IL-1β and Caspase-1 protein expression in sciatic nerve. (B-E) Protein levels were quantified via densitometric analysis of immunoreactive bands in western blots using ImageJ software and were normalized to that of GAPDH. (F-G) Flow cytometry analysis and NLRP3 quantification on CD4 + T cells in splenic MNCs of control (unimmunized group), vehicle and BBG-treated rats. (H) Immunofluorescence photomicrographs of NLRP3 expression in CD4 + T cells in the sciatic nerves of control, vehicle, and BBG-treated EAN rats. (I) Number of NLRP3-positive cells in sciatic nerve sections obtained from control rats, vehicle and BBG-treated EAN rats. (J) NLRP3 quantification on CD4 + T cells. Scale bars, 20 μm. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. Control group, unimmunized group; BBG-P, preventative BBG group; BBG-T, therapeutic BBG group. * p < 0.05, ** p < 0.01

Article Snippet: Following fixation and permeabilization, the cells were further stained with rabbit anti-human P2X7R antibody (Boster Biological Technology) or rabbit anti-human NLRP3 antibody (Thermo Fisher) for 40 min at room temperature, followed by incubation with FITC-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific) for an additional 40 min at room temperature.

Techniques: Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Software, Control