Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: (A) Immortalized GEC that had been depleted of P2X 4 or P2X 7 using lentiviral particles were treated with 100 µM or 3 mM ATP for 3 hours, and supernatants were collected for caspase-1 activity measurement. Caspase-1 activity was measured by ELISA as described in . (B) Total proteins isolated from GEC were subjected to immunoprecipitation (IP) by a polyclonal anti-P2X 4 antibody or Dynabeads as a control. Precipitates or total protein extract (as input) were resolved on SDS-PAGE and analyzed on immunoblots with anti-P2X 7 (top), anti-pannexin-1 (middle), or anti-P2X 4 (bottom) antibodies.

Article Snippet: Antibodies against P2X 4 (APR-002) and P2X 7 (APR-008) were obtained from Alomone Labs.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Isolation, Immunoprecipitation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: Primary GEC (C and D) and immortalized GEC (A and B) were infected with or without P. gingivalis ( P.g. ) at an M.O.I. of 100 for 6 hours, followed by treatment with different pharmaceutical agents. Infected cells were treated with 100 µM ATP, 3 mM ATP, 3 mM ADP, 3 mM AMP, or 3 mM UTP individually for 1 hour (A and C). Alternatively, infected cells were pre-treated with 50 µM 5-BDBD for 15 minutes, 100 µM PPADS for 15 minutes, 100 µM oxATP for 30 minutes, or 1 mM probenecid for 10 minutes, followed by treatment with 3 mM ATP for 1 hour (B and D). The supernatants were collected and subjected to ELISA to measure IL-1β secretion. (E) Primary GEC were transfected with siRNA sequences against P2X 4 or P2X 7 for one day, and mRNA levels were detected by qPCR. (F) Primary GEC depleted of P2X 4 or P2X 7 were infected with P. gingivalis ( P.g. ) and treated with probenecid and 3 mM ATP as shown in (B). IL-1β secretion in the supernatants was analyzed by ELISA. The values showed averages and SD from duplicate samples, which were obtained from three separate experiments.

Article Snippet: Antibodies against P2X 4 (APR-002) and P2X 7 (APR-008) were obtained from Alomone Labs.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Transfection

Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: Model showing the role of P2X 4 and P2X 7 in ROS production and inflammasome activation in GEC stimulated with extracellular ATP.

Article Snippet: Antibodies against P2X 4 (APR-002) and P2X 7 (APR-008) were obtained from Alomone Labs.

Techniques: Activation Assay

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: P2X 7 immunoreactivity in mouse normal skin (A), papilloma (C), and skin cancer tissues (E) (Experiment-1); B, D, F are parallel cross sections, respectively, stained by H&E . G . Analysis of P2X 7 immunoreactivity compared among paired histologically normal and cancerous tissues. Bars are means (± SD) of levels in tissues of five mice. H . P2X 7 protein assays in mouse normal and cancer skin tissues. Lysates fractionated by gel electrophoresis were immunoblotted with the anti P2X 7 antibody and membranes were reprobed with the anti GAPDH antibody. Insert in H shows Western immunoblot of lysates of histologically normal and cancerous tissues obtained from the same animal. Similar results were obtained in tissues of two additional mice. Bars show means (± SD) of densitometry results of the P2X 7 -specific 75 KDa bands in tissues of three mice. I . P2X 7 mRNA levels (relative to GAPDH mRNA) (means ± SD) in histologically normal and cancerous tissues obtained from three animals. In G-I data in the normal tissues were normalized in each case to an arbitrary value of 10. * – p < 0.01. AU – arbitrary units.

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

Techniques: Staining, Nucleic Acid Electrophoresis, Western Blot

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: Effects in mice in-vivo of local treatment with BzATP on skin apoptosis (Experiment-3) . Animals (n = 5) were treated for 16 weeks either with BzATP, applied locally twice a week on the shaved dorsal skin ( A, B ) or with the vehicle (Control, n = 5, C, D ). At the end of the experiment animals were euthanized and strips were obtained from each animal dorsal skin areas for H&E ( B, D ) and P2X 7 /TUNEL co-staining ( E-L ). Arrows in J show increased TUNEL staining co-localizing with P2X 7 immunoreactivity in epidermal cells of the basal/parabasal layers (horizontal arrow) and of epidermal hair shaft cells (vertical arrow). Assays were repeated 3–5 times with similar trends.

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

Techniques: In Vivo, TUNEL Assay, Staining

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: Effects of treatments with BzATP on P2X 7 expression and apoptosis in DMBA/TPA – induced skin papillomas (A-D) and cancers (E-H) (Experiment-1) . Assays were repeated 4 times with similar trends. C, D, G, H are parallel cross sections to A, B, E, F , respectively.

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: Effects of BzATP on apoptosis in cultured mouse keratinocytes . In all experiments levels of apoptosis were normalized to an arbitrary value of 2 in control cells. AU – arbitrary units. A . BzATP dose-response effect in cultured mouse normal (wild-type, C57Bl) keratinocytes (means ± SD, n = 3). Cells were treated with one of the indicated concentrations of BzATP for 8 hours. B . Cultured mouse normal keratinocytes (wild-type, C57Bl) were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Values are means (± SD) of 3 experiments for each condition. Insert in B is Western immunoblot with anti-P2X 7 antibody of lysates of cells treated with ASO or RCO (n = 2). C . BzATP time-response effect in cultured mouse normal keratinocytes (wild-type, C57Bl; filled triangles), or in keratinocytes obtained from P2X 7 -deficient (P2X 7 -/-Pfizer [empty circles] or P2X 7 -/-GSK [filled circles]) mice (both in the C57Bl background). Values are means (± SD) of 3 experiments for each condition. Changes in apoptosis in A and B were determined in terms of solubilized DNA; changes in apoptosis in C were determined using cell-death detection ELISA.

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

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

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: Effects of anti-sense P2X 7 oligonucleotides on BzATP-induced increase in cytosolic calcium (ΔCa 2+ i , A) and on the influx of ethidium bromide (Eth-Br, B) . Cultured mouse wild-type normal keratinocytes were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Levels of ΔCa 2+ i ( A ) and of Eth-Br fluorescence ( B ) were determined as in Fig. 12. The experiments were repeated twice with similar trends.

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

Techniques: Cell Culture, Fluorescence

Journal: BMC Cancer

Article Title: Activation of P2X 7 -mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

doi: 10.1186/1471-2407-9-114

Figure Lengend Snippet: Effects of pre-treatment with anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatments with BzATP (100 μM, 8 hours) on [ 3 H]thymidine incorporation in mouse wild-type normal keratinocytes (values are means ± SD, n = 4) .

Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X 7 receptor antibody, which recognizes the functional full length P2X 7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

Techniques:

Journal: Critical Care

Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

doi: 10.1186/s13054-018-2100-3

Figure Lengend Snippet: Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates P2X receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of P2X 7 can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]

Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

Techniques: Lysis, Concentration Assay, Activation Assay

Journal: Critical Care

Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

doi: 10.1186/s13054-018-2100-3

Figure Lengend Snippet: Freeze-thaw - control experiments. a Whole blood drawn from healthy volunteers was incubated either with vehicle (Veh), 100 μM NF449 (P2X 1 antagonist) or 100 μM A804598 (P2X 7 antagonist) and frozen and stored for 3 weeks as whole blood at − 80 °C. After thawing, lysis was measured as absorbance of free haemoglobin in supernatant at 540 nm. n = 3, mean ± SEM. b Flow cytometry of thawed whole blood samples diluted 1000-fold, to ascertain equal distribution of blood cell sub-populations. Cells were identified by forward scatter FSC and side scatter SSC and quantified based on fixed regions. c Bar graph shows data presented as mean ± SEM, n = 3. ns, not significant

Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

Techniques: Incubation, Lysis, Flow Cytometry

Journal: Critical Care

Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

doi: 10.1186/s13054-018-2100-3

Figure Lengend Snippet: Correlation statistics

Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

Techniques: Activity Assay, Expressing

Journal: Critical Care

Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

doi: 10.1186/s13054-018-2100-3

Figure Lengend Snippet: Haematocrit and haemoglobin levels and erythrocyte P2X 7 receptor expression. a Change in haematocrit in blood pathogen-positive patients with sepsis between Emergency Department (ER) and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows the change in haematocrit in blood pathogen-positive patients grouped by high or low expression of P2X 7 (Δhct/hour). b Change in haematocrit in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. Right panel shows the change in haematocrit in blood pathogen-negative patients grouped by high or low expression of P2X 7 (Δhct/hour). c Change in haemoglobin in blood pathogen-positive patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows change in haemoglobin in blood pathogen-positive patients grouped by high or low expression of P2X 7 (ΔHgb/hour, not significant (ns)). d Change in haemoglobin in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. The right panel shows change in haemoglobin in blood pathogen-negative patients grouped by high or low expression of P2X 7 (ΔHgb/hour)

Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: Expressing, SDS Page, Electrophoresis

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: SDS Page, Electrophoresis, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: Fluorescence, Cell Culture, Staining, Double Staining, Expressing

Journal: Purinergic Signalling

Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment

doi: 10.1007/s11302-016-9541-4

Figure Lengend Snippet: Effect of Ap4A on P2 responses and expression. The effect of Ap4A on ATP-induced calcium rise can be due to a partial agonism effect and/or reduction in receptor expression. Representative traces of calcium determinations after addition of ATP (300 μM) and Ap4A (100 μM) (a, b, and c) show a lower effect of Ap4A, maybe due to a partial agonism on P2X receptors. Western blot analysis of P2X and P2Y receptor expression d showed that Ap4A treatment (100 μM, 24 h) reduces the expression of P2X2, P2X7, P2Y1, and P2Y2 receptors in Neuro-2a cells, while the expression of P2X1, P2X4, and P2Y6 receptors remains unchanged. Panel d shows normalized expression versus the housekeeping protein β-tubulin

Article Snippet: The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control.

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: Specific primers used for RT-PCR studies

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: Qualitative RT-PCR for P2X receptors. ASCs were found to express mRNAs for P2X 3 and P2X 4 receptors. P2X 4 and P2X 7 receptors were upregulated in dASC. However, P2X 6 transcripts were not detected in ASC despite increasing the amount of the starting RNA template. aSC, nSC total RNA were used as positive controls. β -actin was used as a loading control and a reaction omitting the template was used as a negative control

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: P2X 4 and P2X 7 receptor proteins are upregulated in dASC. ( a ) P2X 4 and P2X 7 proteins were not detected in uASC by western blot analysis. Both P2X 4 and P2X 7 receptor proteins were upregulated in dASC to levels comparable to aSC and nSC. The housekeeping gene β -tubulin was used to verify equal loading. ( b – g ) Staining for P2X 4 receptors is faint in uASC ( b ) and strongly increased in dASC ( c ), with a pattern similar to nSC ( d ). Similarly, uASC showed poor positive staining for P2X 7 receptor ( e ), but staining was increased in dASC ( f ) becoming comparable to nSC ( g ). P2X receptors are stained in green and nuclei are stained with DAPI (4′,6- diamidino-2-phenylindole). Negative controls with omission of primary antibodies were performed for each cell type and showed no staining (data not shown)

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Western Blot, Staining

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: Intracellular Ca 2+ signalling induced by stimulation with ATP. ( a – c and g ) uASC ( a ) and dASC ( b ) showed a dose-dependent increase of intracellular Ca 2+ concentration following exposure to ATP, as measured by Fura-2 fluorescence ( n =3). uASC and dASC showed a different ATP sensitivity ( c ), as shown from the quantified AUCs normalised for the maximal response. Intracellular Ca 2+ increase following ATP treatment (1 mM) was also confirmed by confocal imaging of dASC cultures stained with Fluo-4 ( g ). ( d – f ) P2Y contribution to intracellular Ca 2+ increase was assessed by performing Ca 2+ recordings in Ca 2+ -free extracellular solutions; uASC ( d ) and dASC ( e ) showed a different pattern of responses, which saturated at different ATP concentrations ( f ) n =3. ( h and i ) In dASC ( i ), incubation with A10606120 dihydrochloride (300 nM), a potent and specific P2X 7 antagonist, significantly reduced the intracellular Ca 2+ increase evoked by ATP treatments ( n =4, ** P <0.01). This was not observed in uASC ( h ). Statistical analysis was performed using unpaired t -test. Treatments with drug vehicle did not induce any fluorescence changes

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Concentration Assay, Fluorescence, Imaging, Staining, Incubation

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: P2X 7 ion currents in dASCs. ( a ) Representative recordings of ion currents measured from dASC in response to application of increasing concentrations of ATP (upper traces) and BzATP (lower traces); agonists were applied for 30 s with 60-s intervals. ( b ) The concentration dependence of peak amplitude of ion currents recorded as in ( a ); n =6–10 for ATP and 5–10 for BzATP. ( c and d ) Inhibition of ATP-induced ion currents by P2X 7 antagonist AZ 10606120; ATP was applied at 3 mM for 30 s; AZ 10606120 at 300 nM was added to the bath 1–2 min before ATP challenge and remained in the presence of ATP; the average values for peak amplitudes in control and in the presence of the antagonist are shown in ( d ). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n =7, * P <0.05

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Concentration Assay, Inhibition

Journal: Cell Death & Disease

Article Title: Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

doi: 10.1038/cddis.2013.268

Figure Lengend Snippet: P2X 7 activation mediates dASC cell death. ( a ) After 1 h incubation with 5 mM of ATP, cells acquired a rounded morphology typical of dying cells. Cell death was prevented by preincubation with the specific P2X 7 antagonist AZ 10606120 dihydrochloride (300 nM), as shown by bright field images. NT, non-treated controls. ( b ) LDH assay was used to measure cytotoxicity following ATP (1–10 mM) treatments, and a significant increase of cell death was observed only at 5 and 10 mM ATP. ( c ) AZ 10606120 dihydrochloride significantly reduced the ATP-induced cytotoxicity to levels comparable to the controls. Data were normalised to the LDH levels of Triton-X lysates and expressed as percentage of cytotoxicity±S.E.M. ( d ) An MTS assay was performed to measure the cell viability ATP treatment significantly reduced cell viability compared with the NT controls. Pretreatment with AZ 10606120 dihydrochloride prevented the ATP-dependent decrease in cell survival restoring cell viability to levels comparable to NT samples. ( e ) P2X 7 -dependent ATP-induced cell death was further confirmed with EthD-1 staining. Following ATP treatments, the number of death cell stained by EthD-1 was significantly increased. This was prevented by incubation with the AZ 10606120 dihydrochloride compound. For all assays, statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n =6, ** P <0.01, *** P <0.001 and **** P <0.0001)

Article Snippet: The following primary antibodies were applied overnight at 4 °C for P2X 4 (rabbit polyclonal 1 : 1000; Alomone), P2X 7 (rabbit polyclonal 1 : 1000; Alomone) and GFAP (mouse monoclonal 1 : 500; Thermo Scientific).

Techniques: Activation Assay, Incubation, Lactate Dehydrogenase Assay, MTS Assay, Staining