Journal: Biomolecules

Article Title: Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats

doi: 10.3390/biom14020204

Figure Lengend Snippet: Age-dependent fluctuations in P2X7R expression on the plasma membrane in the OFC of wild type and S286L-TG. The left side panel indicates expression of P2X7R in the plasma membrane fraction of the OFC among 4 and 7 weeks of age of wild type (brown columns) and S286L-TG (green columns) were indicated, respectively. Ordinates indicate the mean ± SD ( n = 6) of the relative expression of P2X7R per GAPDH, and abscissas indicate ages (weeks). * p < 0.05, relative to P2X7R expression of 4-week-old S286L, @@ p < 0.01, relative to the 8-week-old wild type using two-way ANOVA with Scheffe’s post hoc test. F values were [F genotype (1,20) = 16.6 ( p < 0.01), F age (1,20) = 2.0 ( p > 0.1), F genotype×age (1,20) = 5.4 ( p < 0.05)]. The right side panel indicates the pseudo-gel images of P2X7R and GAPDH, obtained using capillary immunoblotting. Original Western Blotting Figures can be found in .

Article Snippet: Antibodies against GAPDH (NB300–322, 1:100; Novus Biologicals, Littleton, CO, USA), P2X7R (APR-004,10 μg/mL; Alomone, Jerusalem, Israel), connexin 43 (C6219, 1:100; Sigma-Aldrich), Erk (AF1576, 10 μg/mL; R&D Systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL; R&D Systems), Akt (AF1775, 1 μg/mL; R&D Systems) and phosphorylated Akt (AF877, 5 μg/mL; R&D Systems) were diluted in Immuno Shot Platinum (CosmoBio, Tokyo, Japan).

Techniques: Expressing, Membrane, Western Blot

Journal: Biomolecules

Article Title: Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats

doi: 10.3390/biom14020204

Figure Lengend Snippet: Effects of long-term exposure to BzATP on the expression of P2X7R and connexin 43 and signaling of Akt and Erk in the cultured astrocytes. Interactions among long-term (for 14 d: DIV14-28) exposures to 1 μM BzATP, 10 μM 10-DEBC (Akt inhibitor) and 20 μM FR180204 (Erk inhibitor) on the expression of P2X7R and connexin 43 in the plasma membrane fractions of cultured astrocytes are indicated in panels ( A , B ), respectively. The effects of chronic exposure to 1 μM BzATP on pAkt and pErk are indicated in panels ( C , D ), respectively. Ordinates indicate the mean ± SD ( n = 6) of the relative expression of P2X7R per GAPDH, connexin 43 per GAPDH, pAkt per Akt or pErk per Erk. ** p < 0.01 relative to the expression of the control, and @ p < 0.05, @@ p < 0.01, relative to long-term exposure to 1 μM BzATP alone using one-way ANOVA with Scheffe’s post hoc test. F values of the expression of P2X7R and connexin 43 were [F(3,20) = 16.6 ( p < 0.01)] and [F(3,20) = 19.1 ( p < 0.01)], respectively. F values of the expression of pAkt and pErk were [F(2,15) = 8.2 ( p < 0.01)] and [F(2,15) = 10.9 ( p < 0.01)], respectively. The lower side panels indicate the pseudo-gel images of P2X7R, connexin 43, pAkt and pErk, obtained using capillary immunoblotting. Original Western Blotting Figures can be found in .

Article Snippet: Antibodies against GAPDH (NB300–322, 1:100; Novus Biologicals, Littleton, CO, USA), P2X7R (APR-004,10 μg/mL; Alomone, Jerusalem, Israel), connexin 43 (C6219, 1:100; Sigma-Aldrich), Erk (AF1576, 10 μg/mL; R&D Systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL; R&D Systems), Akt (AF1775, 1 μg/mL; R&D Systems) and phosphorylated Akt (AF877, 5 μg/mL; R&D Systems) were diluted in Immuno Shot Platinum (CosmoBio, Tokyo, Japan).

Techniques: Expressing, Cell Culture, Membrane, Control, Western Blot

Journal: Biomolecules

Article Title: Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats

doi: 10.3390/biom14020204

Figure Lengend Snippet: Time-dependent effects of long-term exposure to BzATP on HFO-evoked ATP release and P2X7R expression in the cultured astrocytes. Astrocytes were incubated in fDMEM without or with 1 μM BzATP for 14 d (DIV14-28). During DIV25-28, astrocytes also received HFO-evoked stimulations (ripple- or fast ripple-evoked stimulations) for 3 d. After the washout, cultured astrocytes were incubated in ACSF without (“non”) or with 1 μM JNJ47965567 (blue columns) or 20 μM Gap19 (green columns), and they then received HFO-evoked stimulation again. Panels ( A , B ) indicate the HFO-evoked ATP releases from cultured astrocytes incubated in fDMEM without or with 1 μM BzATP for 14 d, respectively. Ordinates indicate the mean ± SD ( n = 6) of extracellular ATP levels during HFO-evoked stimulation (nM). @ p < 0.05, @@ p < 0.01 relative to non HFO-evoked stimulation, * p < 0.05, ** p < 0.01 relative to the control using two-way ANOVA with Scheffe’s post hoc test. F values of long-term exposure without and with 1 μM BzATP for 14 d were [F HFO (2,45) = 10.6 ( p < 0.01), F agent (2,45) = 78.9 ( p < 0.01), F agent×HFO (4,45) = 7.7 ( p < 0.01)] and [F HFO (2,45) = 45.7 ( p < 0.01), F agent (2,45) = 159.5 ( p < 0.01), F agent×HFO (4,45) = 19.8 ( p < 0.01)], respectively. Panels ( C , D ) indicate the effects of long-term exposure without and with 1 μM BzATP for 14 d (DIV14-28) with HFO-evoked simulation for 3 d (DIV25-28) on P2X7R expression in the plasma membrane fractions of cultured astrocytes. The ordinate indicates the mean ± SD ( n = 6) of the relative expression of P2X7R per GAPDH. * p < 0.05, ** p < 0.01 relative to the expression of the control, using one-way ANOVA with Scheffe’s post hoc test. F values of long-term exposure without and with 1 μM BzATP for 14 d were [F(2,15) = 5.2 ( p < 0.05)] and [F(2,15) = 24.6 ( p < 0.01)], respectively. The right side panel indicates the pseudo-gel images of P2X7R, obtained using capillary immunoblotting. Original Western Blotting Figures can be found in .

Article Snippet: Antibodies against GAPDH (NB300–322, 1:100; Novus Biologicals, Littleton, CO, USA), P2X7R (APR-004,10 μg/mL; Alomone, Jerusalem, Israel), connexin 43 (C6219, 1:100; Sigma-Aldrich), Erk (AF1576, 10 μg/mL; R&D Systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL; R&D Systems), Akt (AF1775, 1 μg/mL; R&D Systems) and phosphorylated Akt (AF877, 5 μg/mL; R&D Systems) were diluted in Immuno Shot Platinum (CosmoBio, Tokyo, Japan).

Techniques: Expressing, Cell Culture, Incubation, Control, Membrane, Western Blot

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: P2X7R is functional in 4T1 mouse mammary cancer cells and drives cell invasiveness. ( a ) RT-PCR analysis of P2X mRNA expression. ( b ) Representative whole-cell patch clamp recordings of ATP-induced currents. Membrane potential was held at −60 mV. While 10 s application of 10 µM ATP (left) evoked no detec current, application of 5 mM ATP produced a non-desensitizing current that was reduced by treatment with 10 µM A438079 (right). ( c ) Recordings of inward currents from one cell in response to 10 s applications of increasing concentrations of ATP (0.3, 1, 3, 5 and 10 mM) (left), and mean ATP dose-response curve, with currents expressed as a ratio of the maximum current obtained with 10 mM ATP (right). The solid line shows fit to Hill equation. ( d ) Changes in intracellular Ca 2+ levels, indicated by the ratio of Fura2 fluorescence intensity induced by excitation at 340 and 380 nm (F 340/380 ), by exposure to 300 µM BzATP in cells under control conditions (black traces) and cells treated with 10 µM A438079 (red traces) in extracellular Ca 2+ -containing solutions (left) or Ca 2+ -free solutions (right). These data are averaged from four independent experiments. There is statistically significant difference in BzATP-induced Ca 2+ responses between control and A438079-treated cells in the presence of extracellular Ca 2+ at p < 0.001, but no significant difference in the absence of extracellular Ca 2+ , using two-way ANOVA and Dunn’s Multiple Comparison post hoc test. ( e ) BzATP-induced Ca 2+ responses in control cells (vehicle, black traces) and cells treated with 300 nM AZ10606120 (blue traces) in the presence of 3 mM extracellular Ca 2+ (left) or in the absence of extracellular Ca 2+ (right), using similar protocols as in d. These data are averaged from three independent experiments. There is a statistically significant difference in BzATP-induced Ca 2+ responses between control and A438079-treated cells in the presence of 3 mM extracellular Ca 2+ at p < 0.001, but no significant difference in the absence of extracellular Ca 2+ , using two-way ANOVA and Dunn’s Multiple Comparison post hoc test. ( f ) Cell viability after exposure to increasing concentrations of ATP (from 0.03 to 3 mM) for 24 h without or with treatment with 10 µM A438079, expressed as % of that under control conditions (vehicle). Results are from four independent experiments. ATP induced a statistically significant cell viability reduction compared to control conditions at * p < 0.05, ** p < 0.01 and *** p < 0.0001, but A438079 treatment had no significant effect on ATP-induced reduction in cell viability, using Student’s t -test. ( g ) Cell viability after exposure to increasing concentrations of BzATP (from 0.01 to 1 mM) for 24 h, expressed as % of that under control condition (vehicle). Results are from four independent experiments. BzATP induced no significant reduction in cell viability. ( h ), 4T1 cell invasiveness, assessed using trans-well invasion inserts after 24 h in the absence or presence of 300 µM BzATP, in cells transfected with control siRNA (siCTL) or P2X7R-specific siRNA (siP2X7). Results from six independent experiments expressed relative to the siCTL condition, in the absence of BzATP stimulation (CTL). ** indicates a statistically significant difference at p < 0.01 when comparing siCTL/BzATP to siCTL/CTL, using Mann-Whitney Rank Sum Test. NS stands for no statistical difference. ( i ) 4T1 2D-cell invasiveness was assessed over 24 h using trans-well invasion inserts in the absence or presence of BzATP (300 µM), and in the absence or presence of P2X7 antagonist A438079 (10 µM). Results are from 4–6 independent experiments and are expressed relatively to the control condition (CTL), in the absence of BzATP and of A438079. * indicates a statistically significant difference at p < 0.05 when comparing BzATP treatment to CTL, using Wilcoxon Signed Rank Test. ( j ) 4T1 3D-cell invasiveness was assessed over 72 h. The left image shows cancer cell spheroid morphology assessed before (T = 0 h) or after treatment (T = 72 h) in control condition (vehicle) or with BzATP (300 µM) in the absence or presence of A438079 (10 µM). Scale bar, 100 µm. On the right side is the summary of assessment of the spheroid circularity from 14–20 independent experiments. * indicates a statistically significant difference at p < 0.05 using Dunn’s Multiple Comparison Test.

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Patch Clamp, Membrane, Produced, Fluorescence, Control, Comparison, Transfection, MANN-WHITNEY

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: P2X7 receptor promotes invadopodial activity of extracellular matrix degradation. ( a ) 4T1 mouse mammary and MDA-MB-435s human cancer cells were grown on Matrigel™ containing DQ-BSA as a fluorogenic substrate for proteases, emitting red fluorescence when degraded. P2X7 receptor was immunodetected using a rabbit primary antibody against P2X7 protein and a secondary ant-rabbit IgG antibody conjugated to AF488. The right panel shows the fluorescence values for the two channels at the locations indicated by a blue arrow. Scale bar, 100 µm. ( b ) Invadopodia entrapped into a 2%-gelatin matrix were fractionated and separated from cytosol and membranes-enriched fractions. The quality of the fractions was assessed by western blotting after SDS-PAGE, using cytosolic (HSC70), membrane (caveolin-1, β-adaptin) and invadopodia (cathepsin B, cortactin, focal adhesion kinase) markers. P2X7 proteins were found to be present in the membrane fractions and enriched in the invadopodia fraction. This figure is representative of 5 independent experiments. The full image blots can be found in . ( c ) The invadopodial activity was assessed as being F-actin foci (green labelling, phalloidin-488) co-localised with focused proteolytic activities (red labelling, DQ-BSA proteolysis) from 4T1 cells grown for 24 h on Matrigel™ containing DQ-BSA in control condition (vehicle), or stimulated with 300 µM BzATP, in the presence and absence of 10 µM A438079. “Coloc” indicates co-localization of degradative activity and F-actin foci and appears as white pixels. Scale bar, 50 µm. ( d ) The number of degradation areas per cell was counted in 20 images, from five independent experiments. * indicates a statistically significant difference at p < 0.05 using Dunn’s Multiple Comparison Test. NS stands for no statistical difference. ( e ) The number of invadopodia (identified as being F-actin foci colocalized with matrix degradation spots) was counted per cell in the same experiments as in d. There was no significant difference among the four treatment conditions. ( f ) Western blot analyses for cathepsin B (Cath B) in concentrated cell-free supernatants of 4T1 cells after 24 h treatment with BzATP (300 µM) in presence or not 10 µM A438079. Pro- and mature forms of Cath B were detected (top). β-actin was detected in corresponding cell lysates and was used as an internal control. Amounts of released mature forms of Cath B in supernatants of 4T1 cells in the three experimental conditions were analysed, from five independent experiments and expressed as a proportion of β-actin in corresponding cells. Box plots indicate median, first and third quartile and the mean is indicated as a square. * indicates a statistically significant difference at p < 0.05 using Dunn’s Multiple Comparison Test. The full image blots can be found in .

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: Activity Assay, Fluorescence, Western Blot, SDS Page, Membrane, Control, Comparison

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: P2X7 receptor stimulation promotes the acquisition of a mesenchymal phenotype. ( a ) F-actin cytoskeleton was visualized using phalloidin-488 in 4T1 cells stimulated with 300 µM BzATP at the time indicated. Rapid morphological changes appear as rapidly as after 6 min stimulation. Scale bar, 25 µm. ( b ) The number of new filopodia per cell was counted per hour in cells transfected with the LifeAct plasmid and studied in the presence of 300 µM BzATP, in the presence or absence of 10 µM A438079. * indicates a statistically significant difference at p < 0.05 using Dunnett’s Multiple Comparison Test. ( c ) The velocity of filopodia to form and extend (in µm/min) was measured in the same experiments as in ( b ). There was no significant difference among the four experimental groups. ( d ) On the left side, representative western blots show total and active GTP-bound forms of Cdc42, RhoA, and Rac1 pulled down by GST-RBD in 4T1 cells in control conditions or stimulated for 30 min with 300 µM BzATP, in the presence and absence of 10 µM A438079. His-tagged proteins are used as positive controls for the quality of the pull-down experiment. On the right side, graphs show quantifications of GTP-bound RhoGTPases (active form), normalized to total protein level, and expressed relatively to that of the control condition (vehicle). Results are from 4–7 independent experiments. * indicates statistically significant difference at p < 0.05, using Mann-Whitney rank sum test. NS stands for no statistical difference. The full image blots can be found in . ( e ) mRNA expression levels of P2rx7 , SNAI1 , ZEB1 , TWIST1 , ZO1 and CDH1 assessed by RT-qPCR in 4T1 cells stimulated by 300 µM BzATP for 24 h and expressed relatively to the GUSB reference gene and as ratios to the expression levels in control condition without BzATP ( n = 9–11 independent experiments). SNAI1 was significantly higher, and ZO1 was significantly lower after BzATP treatment compared to control condition at * p < 0.05, using Mann-Whitney rank sum test. ( f ) representative western blots (from four independent experiments) show vimentin and E-cadherin expression in 4T1 cells in control conditions or stimulated for 24h with 300 µM BzATP, in the presence and absence of 10 µM A438079. There was no significant effect. The full image blots can be found in .

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: Transfection, Plasmid Preparation, Comparison, Western Blot, Control, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: P2X7 receptor in mammary cancer cells enhances primary tumour growth and metastatic development in vivo. ( a ) P2rx7 mRNA expression levels assessed by RT-qPCR in CTL, Crispr#1 and Crispr#2 cell lines, expressed relatively to 4T1 cells. * indicated a statistically significant difference from CTL cells at p < 0.05 using Mann-Whitney Rank sum test. ( b ) 2-D cancer cell invasiveness of CTL, Crispr#1 and Crispr#2 cell lines, in the absence or presence of 300 µM BzATP, expressed relatively to the CTL cell line without BzATP ( n = 5–8 independent experiments). * indicates a statistically significant difference from CTL/no BzATP condition at p < 0.05, ¤ indicates a statistically significant difference from CTL/BzATP at p < 0.05, using Dunn’s Multiple Comparison Test. NS, stands for no statistically significant difference. ( c ) Cell proliferation over 4 days of CTL, Crispr#1 and Crispr#2 cell lines in the absence of 300 µM BzATP, expressed relatively to the CTL condition. Results are from 6–10 independent experiments. * indicates a statistically significant difference from CTL at p < 0.05 using Dunn’s Multiple Comparison Test. ( d ) Cell adhesion of CTL, Crispr#1 and Crispr#2 clones, expressed relatively to CTL. Results are from 10 independent experiments. * indicates a statistically significant difference from CTL at p < 0.05 using Dunn’s Multiple Comparison Test. ( e–i ) In vivo mouse experiments testing the effect of P2rx7 gene expression in host mice on primary tumour growth and metastasis development, with two numbers of cancer cells implanted. ( e ) Top, cartoon describing the experimental procedure, in which CTL cells, derived from 4T1-Luc expressing P2rx7 gene, were implanted to the fifth mammary fat pad of 6 weeks-old female wild-type BALB/cJ ( P2rx7 + / + ) or P2X7 knock-out BALB/cJ (P2rx7 − / − ) mice. Bottom, representative bioluminescent images of a wild-type mouse, on 14 and 35 days after cell implantation, identifying primary tumours as well as metastatic foci. ( f ) Primary mammary tumour growth in wild-type BALB/cJ ( P2rx7 + / + , n = 8) or P2X7 knock-out BALB/cJ (P2rx7 − / − , n = 8) mice as a function of time following the implantation of 1 × 10 6 CTL cells on day 0. ( g ) Analysis of the number of metastases per mouse, identified at necropsy, in the experiment described in ( f ). NS stands for no statistical significant difference. ( h ) Primary mammary tumour growth in wild-type BALB/cJ ( P2rx7 + / + , n = 8) or P2X7 knock-out BALB/cJ ( P2rx7 − / − , n = 8) mice as a function of time following the implantation of 1 × 10 4 CTL cells on day 0. ( i ) Analysis of the number of metastases per mouse, identified at necropsy, in the experiment described in ( h ). NS stands for no statistically significant difference. ( j–l ) In vivo mouse experiments assessing the role of P2rx7 gene expression in mammary cancer cells on primary tumour growth and metastasis development. ( j ) Cartoon describing the experimental procedure, in which CTL ( P2rx7 + / + ), Crispr#1 or Crispr#2 (both knock-down for the P2rx7 expression) cells were implanted to the fifth mammary fat pad of 6 weeks-old female wild-type BALB/cJ ( P2rx7 + / + ) at a density of 1 × 10 4 CTL cells per mouse (8 mice per group). ( k ) Primary mammary tumour growth in wild-type BALB/cJ ( P2rx7 + / + ) as a function of time following the implantation of CTL, Crispr#1 or Crispr#2 cells on day 0. *** indicates a statistically significant difference from the CTL group at p < 0.001 using two-way ANOVA. ( l ) Analysis of the number of metastases per mouse, in the three experimental groups, identified at necropsy, in the experiment described in ( j ). * indicates a statistically significant difference from the CTL group at p < 0.05 using Dunn’s Multiple Comparison test.

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, CRISPR, MANN-WHITNEY, Comparison, Clone Assay, Gene Expression, Derivative Assay, Knock-Out, Knockdown

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: P2X7 antagonism slows mammary tumour growth. ( a ) In vivo mouse experiments assessing the effects of treatments with two P2X7 antagonist (A438079 or AZ10606120) on primary tumour growth, in which cancer cells and host organisms both express the P2rx7 gene. ( b ) primary mammary tumour growth represented as a function of the duration of the treatment depending on antagonist treatments (A438079 or AZ10606120) compared to the injection of vehicle. ( c – e ) A Gompertz model was used to assess mammary tumour growth as a function of time for ( c ), vehicle group, ( d ), A438079 and ( e ), AZ10606120. Black, red and blue dots represent individual tumour volumes of these respective categories, blue areas are 90% model prediction intervals and lines are the median tumour growth. Estimated mean (inter-individual standard deviation) of model parameters were k growth = 0.64 day-1 (39%), V max = 1.620 mm 3 (64%), EFF = 0.52 (82%) and γ = 0.17 (–). This analysis showed that tumour volume doubled in a mean time of 0.64 day, that the mean maximum tumour volume was 1.620 mm 3 and that tumour growth was twice slower in the presence of A438079 or AZ10606120 treatment ( p < 10 −10 ) as compared to the vehicle group. There was no difference in the efficacy of these two treatments ( p = 0.08). ( f ) Assessment of the number of metastases per mouse, in the three experimental groups; vehicle, A438079 and AZ10606120. There was no statistical difference. ( g ) Percent survival curve in the three experimental group indicating a significant prolonged survival of mice treated with AZ10606120 at p = 0.0464 using Gehan-Breslow-Wilcoxon Test.

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: In Vivo, Injection, Standard Deviation

Journal: Cancers

Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

doi: 10.3390/cancers12092342

Figure Lengend Snippet: Sequences of conventional Polymerase Chain Reactions (PCR) and quantitative Polymerase Chain Reactions (qPCR) primers and expected amplicon size.

Article Snippet: Primary antibodies used were: mouse anti-HSC70 (sc-7298 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), rabbit anti-caveolin 1 (sc-894 Santa Cruz Biotechnology, Inc., Germany), rabbit anti-β-adaptin (610382 BD Biosciences, Le Pont de Claix, France), cathepsin B (20-CR71 Fitzgerald, Acton, MA, USA), cortactin (05-180 Millipore, Guyancourt, France), FAK (036SC-558 Santa Cruz Biotechnology, Inc., Heidelberg, Germany), P2X7 (APR-008 Alomone Labs Ltd., Jerusalem, Israel).

Techniques: Amplification

Journal: iScience

Article Title: Hepatitis B virus-mediated sodium influx contributes to hepatic inflammation via synergism with intrahepatic danger signals

doi: 10.1016/j.isci.2023.108723

Figure Lengend Snippet:

Article Snippet: anti-P2X7 , Cell signaling Techonology , 13809.

Techniques: Activity Assay, ATP Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Plasmid Preparation, RNA Sequencing Assay, Biomarker Assay, Software

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 1 | Molecular docking of dihydromyricetin (DHM) on P2X7 receptor. (A) Simulation modeling of DHM docking on P2X7 receptor was performed by a computer. Molecular docking prediction of DHM on P2X7 receptor was performed by AutoDock 4.2. (B–D) Enlarged view indicating the perfect match enabling DHM to interact with P2X7 receptor.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 4 | Effects of dihydromyricetin (DHM) on the expression of P2X7 mRNA in the dorsal root ganglia (DRGs; A), spinal cord (B), and hippocampus (C) of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Effects of DHM on the expression of P2X7 protein in the dorsal root ganglia (DRGs; D), spinal cord (E), and hippocampus (F) of rats with model. Data are displayed as the means ± standard error of the mean. **p<0.01 vs. control group;

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Expressing, Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 5 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the dorsal root ganglia (DRGs) of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 100 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 6 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the spinal cord of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 100 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 7 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the hippocampus of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 200 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control