Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: P2X7 antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: EOC13 microglial cells express P2X7. (a) RNA from EOC13 and J774 cells was amplified by RT-PCR using primers for P2X7. Water in place of RNA was included as a negative control in the PCR reaction. PCR products were separated and visualised with ethidium bromide staining. (b) EOC13 and J774 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-P2X7 Ab. (c) EOC13 and J774 cells were labelled with an anti-P2X7 (solid line) or isotype control (shaded) mAb and then with APC-conjugated anti-IgG Ab and 7AAD (to exclude dead cells). Relative P2X7 expression (mean fluorescence intensity) was determined by flow cytometry. (d) Fixed and permeabilised EOC13 and J774 cells were labelled with an anti-P2X7 Ab and then with Cy3-conjugated anti-IgG Ab. P2X7 (top panels) and phase contrast (bottom panels) images were assessed by confocal microscopy. Bars represent 10 μ m. (a–d) Results are representative of 2-3 experiments.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Staining, SDS Page, Expressing, Fluorescence, Flow Cytometry, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: EOC13 microglial cells express functional P2X7. (a and b) EOC13 cells in NaCl medium were incubated with 25 μ M ethidium + in the absence (basal) or presence of (a) 1 mM ATP, 0.1 mM BzATP, or (b) varying concentrations of ATP (as indicated) at 37°C for 5 min. (c and d) Cells in NaCl medium were preincubated in the absence (control) or presence of (c) 30 μ M Brilliant Blue G (BBG), 100 μ M A438079, 30 μ M AZ11645373, or (c and d) 10 μ M AZ10606120 at 37°C for 15 min. (c) Ethidium + (25 μ M) or (d) YO-PRO-1 2+ (1 μ M) was then added, and (c and d) cells were incubated in the absence (basal) or presence of 1 mM ATP at 37°C for 5 min. (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a, c, and d) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of antagonist. (b) Curve presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Functional Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: P2X7 activation induces ROS formation in EOC13 microglial cells. (Left panels) Adherent DCF-loaded EOC13 cells or (right panels) suspended EOC13 cells in (a) NaCl medium containing 1 mM Ca 2+ (preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min), (b) NaCl medium in the absence (control) or presence of 1 mM Ca 2+ , (c) NaCl medium in the absence (control) or presence of 100 μ M EGTA, or (d) NaCl or KCl medium were (a–d) incubated in the absence (basal) or presence of 575 μ M ATP 4− (2 mM or 1.4 mM ATP as explained in Section 2.8) at 37°C for (left panels) 15 min or (right panels) 5 min in the presence of 25 μ M ethidium + . (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of (left panels) DCF (ROS formation) or (right panels) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 or ** P < 0.01 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: The ROS scavenger NAC inhibits P2X7-induced ROS and pore formation in EOC13 microglial cells. (a and c) Adherent DCF-loaded EOC13 cells or (b) suspended EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 40 mM NAC at 37°C for 30 min and then in the absence (basal) or presence of 1.4 mM ATP for (a and c) 15 min or (b) 5 min in the presence of 25 μ M ethidium + . (a–c) Incubations were stopped by the addition of MgCl 2 medium and (a and b) centrifugation. (a and b) Mean fluorescence intensities (MFI) of (a) DCF (ROS formation) or (b) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of NAC. (c) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. Results are representative of 2 experiments.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Centrifugation, Fluorescence, Flow Cytometry, Microscopy

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: P2X7 activation induces NO formation in EOC13 microglial cells. Adherent DAF-FM DA-loaded EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 1.4 mM ATP for 15 min. Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of benzotriazole (NO formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Centrifugation, Fluorescence, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P < 0.001 or * P < 0.05 compared to (a and c) 0 mM ATP, (b and d) corresponding basal, or (e) corresponding 0 min ATP; ††† P < 0.001 compared to corresponding ATP in the absence of (b and d) AZ10606120 or (e) NAC.

Article Snippet: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Incubation, Flow Cytometry, Microscopy, Centrifugation, Fluorescence

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Fluorescence, Staining

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: MANN-WHITNEY, Activation Assay