anti p2x7 receptor antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti p2x7 receptor antibody
    <t>P2X7</t> receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P
    Anti P2x7 Receptor Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 96/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x7 receptor antibody/product/Alomone Labs
    Average 96 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    anti p2x7 receptor antibody - by Bioz Stars, 2022-10
    96/100 stars

    Images

    1) Product Images from "Asymmetric activation of microglia in the hippocampus drives anxiodepressive consequences of trigeminal neuralgia"

    Article Title: Asymmetric activation of microglia in the hippocampus drives anxiodepressive consequences of trigeminal neuralgia

    Journal: bioRxiv

    doi: 10.1101/2022.04.16.488241

    P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P
    Figure Legend Snippet: P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

    Techniques Used: Activation Assay, Western Blot

    2) Product Images from "Heightened inflammasome activation is linked to age-related cognitive impairment in Fischer 344 rats"

    Article Title: Heightened inflammasome activation is linked to age-related cognitive impairment in Fischer 344 rats

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-12-123

    Aging alters expression of NLRP1 inflammasome components . Representative immunoblots and densitometric analysis of immunoblots for caspase-1 (Casp1), caspase-11 (Casp11), NLRP1, ASC, cleaved XIAP, P2X7 receptor (P2X7R) and pannexin-1 (PanX1) in brain lysates of young (Y) and aged (A) animals. Protein levels of cleaved caspase-1 and -11 are higher in aged animals compared to young. Protein levels of NLRP1 and ASC did not change with age whereas P2X7 receptor, the pannexin-1 protein and the cleaved fragment of XIAP are higher in aged animals than in young animals. β-Tubulin was used as an internal standard and control for protein loading. Data are presented as mean +/- SEM. *p
    Figure Legend Snippet: Aging alters expression of NLRP1 inflammasome components . Representative immunoblots and densitometric analysis of immunoblots for caspase-1 (Casp1), caspase-11 (Casp11), NLRP1, ASC, cleaved XIAP, P2X7 receptor (P2X7R) and pannexin-1 (PanX1) in brain lysates of young (Y) and aged (A) animals. Protein levels of cleaved caspase-1 and -11 are higher in aged animals compared to young. Protein levels of NLRP1 and ASC did not change with age whereas P2X7 receptor, the pannexin-1 protein and the cleaved fragment of XIAP are higher in aged animals than in young animals. β-Tubulin was used as an internal standard and control for protein loading. Data are presented as mean +/- SEM. *p

    Techniques Used: Expressing, Western Blot

    Aging induces alterations in protein expression patterns of Caspase-1, pannexin-1 and P2X7R in hippocampal neurons . Confocal images show hippocampal neurons in the CA3 region of young and aged animals. Sections were stained for caspase-1, pannexin-1 and P2X7 (red) and the neuronal marker NeuN (green). In aged animals, the immunoreactivity of caspase-1, P2X7 and pannexin-1 are increased in neurons of the hippocampus compared to young animals with P2X7 and pannexin-1 showing a polarized distribution near the somatic membrane (arrows). Bar=20 μm.
    Figure Legend Snippet: Aging induces alterations in protein expression patterns of Caspase-1, pannexin-1 and P2X7R in hippocampal neurons . Confocal images show hippocampal neurons in the CA3 region of young and aged animals. Sections were stained for caspase-1, pannexin-1 and P2X7 (red) and the neuronal marker NeuN (green). In aged animals, the immunoreactivity of caspase-1, P2X7 and pannexin-1 are increased in neurons of the hippocampus compared to young animals with P2X7 and pannexin-1 showing a polarized distribution near the somatic membrane (arrows). Bar=20 μm.

    Techniques Used: Expressing, Staining, Marker

    Probenecid reduces protein expression of NLRP1 inflammasome and ameliorates spatial learning deficits in aged rats . (A) Representative immunoblots of cleaved caspase-1, pannexin1 and P2X7R in hippocampal lysates of vehicle (Veh)-treated and probenecid (Pr)-treated 18-month-old rats. β-tubulin was used as an internal control. (B) Densitometric analysis of immunoblots from brain lysates of cleaved caspase-1 (Casp1), P2X7 receptor (P2X7R), and pannexin1 (PanX1). (C-D) Aged animals underwent behavioral testing following either probenecid or vehicle treatment. (C) In a hippocampal-dependent spatial learning task via Morris water maze, latency to platform was measured on days 1-3 and 8-10. Probenecid-treatment improved latency to platform measured on the final day of testing (D) Mean path length was determined on day 10 of testing and probenecid-treated rats demonstrated significantly shorter mean path lengths than vehicle-treated controls. Drug treatment was administered twice daily for 3 days (days 7-9). Data are presented as mean +/- SEM *p
    Figure Legend Snippet: Probenecid reduces protein expression of NLRP1 inflammasome and ameliorates spatial learning deficits in aged rats . (A) Representative immunoblots of cleaved caspase-1, pannexin1 and P2X7R in hippocampal lysates of vehicle (Veh)-treated and probenecid (Pr)-treated 18-month-old rats. β-tubulin was used as an internal control. (B) Densitometric analysis of immunoblots from brain lysates of cleaved caspase-1 (Casp1), P2X7 receptor (P2X7R), and pannexin1 (PanX1). (C-D) Aged animals underwent behavioral testing following either probenecid or vehicle treatment. (C) In a hippocampal-dependent spatial learning task via Morris water maze, latency to platform was measured on days 1-3 and 8-10. Probenecid-treatment improved latency to platform measured on the final day of testing (D) Mean path length was determined on day 10 of testing and probenecid-treated rats demonstrated significantly shorter mean path lengths than vehicle-treated controls. Drug treatment was administered twice daily for 3 days (days 7-9). Data are presented as mean +/- SEM *p

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels *"

    Article Title: Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.642033

    The pre-TM1 region regulates hP2X7 facilitation. A , representative traces showing the speeding/facilitation of WT P2X7 receptor responses to prolonged (60 s, bar ) repeat applications of 1 m m ATP at 3-min intervals ( black , red , and blue trace consecutively). B , increasing the time interval between repeat ATP applications 2 and 3 ( black trace to red trace , 3-min interval; red trace to blue trace , 10-min interval) returns the receptor response back to its naïve state. C , schematics and representative traces showing the effect of replacing the entire N terminus of the hP2X7 receptor ( black ) with that of hP2X2 receptor ( magenta ) or just the 16 pre-TM1 amino acids ( P2X7–2N β). Traces represent first ( black ) and second ( red ) receptor responses (60-s application at 3-min intervals) to 1 m m ATP. ex , the extracellular region of the receptor. D , histogram summary showing the 10–50% rise times (seconds) of the first ( black ) versus second ( red ) receptor response to EC 90 ATP (1 m m ATP at P2X7 and 100 μ m ATP at P2X7–2N, P2X7–2Nβ, and P2X2) at 3-min intervals. Inset , concentration response to ATP for P2X7 and P2X7–2Nβ receptors. E , histogram summary showing the peak amplitude (microamperes) of the response to the first ATP application. Inset , representative Western blot of the equivalent levels of surface expression of P2X7 and P2X7–2Nβ receptors. Data are mean ± S.E. ( n = 7–25). *, p
    Figure Legend Snippet: The pre-TM1 region regulates hP2X7 facilitation. A , representative traces showing the speeding/facilitation of WT P2X7 receptor responses to prolonged (60 s, bar ) repeat applications of 1 m m ATP at 3-min intervals ( black , red , and blue trace consecutively). B , increasing the time interval between repeat ATP applications 2 and 3 ( black trace to red trace , 3-min interval; red trace to blue trace , 10-min interval) returns the receptor response back to its naïve state. C , schematics and representative traces showing the effect of replacing the entire N terminus of the hP2X7 receptor ( black ) with that of hP2X2 receptor ( magenta ) or just the 16 pre-TM1 amino acids ( P2X7–2N β). Traces represent first ( black ) and second ( red ) receptor responses (60-s application at 3-min intervals) to 1 m m ATP. ex , the extracellular region of the receptor. D , histogram summary showing the 10–50% rise times (seconds) of the first ( black ) versus second ( red ) receptor response to EC 90 ATP (1 m m ATP at P2X7 and 100 μ m ATP at P2X7–2N, P2X7–2Nβ, and P2X2) at 3-min intervals. Inset , concentration response to ATP for P2X7 and P2X7–2Nβ receptors. E , histogram summary showing the peak amplitude (microamperes) of the response to the first ATP application. Inset , representative Western blot of the equivalent levels of surface expression of P2X7 and P2X7–2Nβ receptors. Data are mean ± S.E. ( n = 7–25). *, p

    Techniques Used: Concentration Assay, Western Blot, Expressing

    Contribution of the hP2X7 receptor C-terminal cysteine-rich region (amino acid residues 362–379) to the time course. A , peak normalized traces showing the effect on the time-course of cysteine-rich region deletion of the WT P2X7 receptor, insertion into the P2X2 receptor, and insertion into the P2X1 receptor. ATP application was as indicated by the gray bars (1 m m for P2X7 and P2X7–1Nβ and 100 μ m for P2X1 and P2X2 WT receptors and chimeras). For the P2X7-delCcys, the trace in yellow shows the relative amplitude of the response compared with the P2X7 receptor to show the similar time course to the initial rise time of the P2X7 receptor response. B—D , histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse. Western blots show equivalent surface expression of the respective WT and chimeric receptors (the WT control for P2X7 is the same as in Fig. 3 , the original blot compared P2X7 WT with a range of P2X7 chimeras). Data are mean ± S.E. ( n = 4–25). *, p
    Figure Legend Snippet: Contribution of the hP2X7 receptor C-terminal cysteine-rich region (amino acid residues 362–379) to the time course. A , peak normalized traces showing the effect on the time-course of cysteine-rich region deletion of the WT P2X7 receptor, insertion into the P2X2 receptor, and insertion into the P2X1 receptor. ATP application was as indicated by the gray bars (1 m m for P2X7 and P2X7–1Nβ and 100 μ m for P2X1 and P2X2 WT receptors and chimeras). For the P2X7-delCcys, the trace in yellow shows the relative amplitude of the response compared with the P2X7 receptor to show the similar time course to the initial rise time of the P2X7 receptor response. B—D , histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse. Western blots show equivalent surface expression of the respective WT and chimeric receptors (the WT control for P2X7 is the same as in Fig. 3 , the original blot compared P2X7 WT with a range of P2X7 chimeras). Data are mean ± S.E. ( n = 4–25). *, p

    Techniques Used: Western Blot, Expressing

    Functional interaction between the pre-TM1 β-region and C-terminal cysteine-rich region and its effect on P2X7 receptor time course. A , peak normalized traces showing the effect of deletion of the cysteine-rich region deletion from the P2X7 receptor (P2XYdelCcys, yellow ) compared with the WT P2X7 receptor ( black ) and the P2X7–2Nβ chimera ( red ). ATP application is indicated by the gray bars (1 m m for 60 s). B , peak normalized traces showing reversion to slow receptor facilitation at the P2X7–2Nβ delCcys chimera ( blue ) with both 60-s ( i ) and 120-s ( ii ) applications of 1 m m ATP highlighting the faster desensitization of the chimeric receptor. C–E , histograms showing the peak amplitude (microamperes), rise time (seconds), and time to 50% decay (seconds) at the end of the ATP pulse for the P2X7 receptor versus the P2X7–2Nβ delCcys chimera. Western blots show equivalent levels of surface expression of P2X7 and the P2X7–2Nβ delCcys chimera. Data are mean ± S.E. ( n = 4–25). ***, p
    Figure Legend Snippet: Functional interaction between the pre-TM1 β-region and C-terminal cysteine-rich region and its effect on P2X7 receptor time course. A , peak normalized traces showing the effect of deletion of the cysteine-rich region deletion from the P2X7 receptor (P2XYdelCcys, yellow ) compared with the WT P2X7 receptor ( black ) and the P2X7–2Nβ chimera ( red ). ATP application is indicated by the gray bars (1 m m for 60 s). B , peak normalized traces showing reversion to slow receptor facilitation at the P2X7–2Nβ delCcys chimera ( blue ) with both 60-s ( i ) and 120-s ( ii ) applications of 1 m m ATP highlighting the faster desensitization of the chimeric receptor. C–E , histograms showing the peak amplitude (microamperes), rise time (seconds), and time to 50% decay (seconds) at the end of the ATP pulse for the P2X7 receptor versus the P2X7–2Nβ delCcys chimera. Western blots show equivalent levels of surface expression of P2X7 and the P2X7–2Nβ delCcys chimera. Data are mean ± S.E. ( n = 4–25). ***, p

    Techniques Used: Functional Assay, Western Blot, Expressing

    Contribution of variant pre-TM1 residues to P2X7 receptor current facilitation. A , amino acid sequence lineup showing the pre-TM1 residues of the hP2X7 receptor ( top row ) and the hP2X2 receptor ( bottom row ). Non-conserved amino acids are shown in green. B , representative traces (the first and second responses are shown in black and red , respectively) demonstrating the effect of pre-TM1 substitution of non-conserved amino acid residues between the P2X7 and P2X2 receptor in response to ATP (1 m m , 60-s agonist addition at 3-min intervals). C and D , histogram summaries showing the peak current amplitude (microamperes and 10–50% rise time (seconds) of the first and second responses to ATP. Statistical significance shown in black is relative to the P2X7 WT and, in red , for between the first and second response at a particular receptor. Data are mean ± S.E. ( n = 4–25). **, p
    Figure Legend Snippet: Contribution of variant pre-TM1 residues to P2X7 receptor current facilitation. A , amino acid sequence lineup showing the pre-TM1 residues of the hP2X7 receptor ( top row ) and the hP2X2 receptor ( bottom row ). Non-conserved amino acids are shown in green. B , representative traces (the first and second responses are shown in black and red , respectively) demonstrating the effect of pre-TM1 substitution of non-conserved amino acid residues between the P2X7 and P2X2 receptor in response to ATP (1 m m , 60-s agonist addition at 3-min intervals). C and D , histogram summaries showing the peak current amplitude (microamperes and 10–50% rise time (seconds) of the first and second responses to ATP. Statistical significance shown in black is relative to the P2X7 WT and, in red , for between the first and second response at a particular receptor. Data are mean ± S.E. ( n = 4–25). **, p

    Techniques Used: Variant Assay, Sequencing

    Interaction of the intracellular pre-TM1 β region and C-terminal cysteine-rich region of the P2X7 receptor regulates pore formation and dye uptake. A , FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor, reduced dye uptake though the N terminus chimeric receptor, and C terminus deletion receptor (P2X7–2Nβ and P2X7 delCcys, respectively) and “P2X7-like” dye uptake through the P2X7–2Nβ delCcys mutant receptor. Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow . S.E. is only shown in one direction to not obscure the mean values. Western blots show equivalent levels of surface expression for the P2X7 receptor WT and mutants. B , P2X7–2Nβ receptor response normalized to the peak P2X7 receptor response identifying an increased initial rate of dye uptake. C , P2X7–2Nβ delCcys receptor response normalized to the peak P2X7 receptor response demonstrating the relative dye uptake speeds through these two receptors. D , histogram summary showing the relative dye uptake for each receptor. E , histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μ m BzATP addition made at 240 s). Data are mean ± S.E. **, p
    Figure Legend Snippet: Interaction of the intracellular pre-TM1 β region and C-terminal cysteine-rich region of the P2X7 receptor regulates pore formation and dye uptake. A , FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor, reduced dye uptake though the N terminus chimeric receptor, and C terminus deletion receptor (P2X7–2Nβ and P2X7 delCcys, respectively) and “P2X7-like” dye uptake through the P2X7–2Nβ delCcys mutant receptor. Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow . S.E. is only shown in one direction to not obscure the mean values. Western blots show equivalent levels of surface expression for the P2X7 receptor WT and mutants. B , P2X7–2Nβ receptor response normalized to the peak P2X7 receptor response identifying an increased initial rate of dye uptake. C , P2X7–2Nβ delCcys receptor response normalized to the peak P2X7 receptor response demonstrating the relative dye uptake speeds through these two receptors. D , histogram summary showing the relative dye uptake for each receptor. E , histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μ m BzATP addition made at 240 s). Data are mean ± S.E. **, p

    Techniques Used: Mutagenesis, Western Blot, Expressing

    Effects of mutations in the P2X7 receptor pre-TM1 β-region on pore formation and dye uptake. A , FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor and reduced dye uptake though the N terminus mutants N16P, Y26L, S23N, and NRRL. Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow . Standard errors are only shown in one direction to not obscure the mean values. B , histogram summary showing the relative peak dye uptake for each receptor. Western blots show equivalent levels of surface receptor expression for P2X7 receptor WT and mutants. C , histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μ m BzATP addition made at 240 s). Data are mean ± S.E. **, p
    Figure Legend Snippet: Effects of mutations in the P2X7 receptor pre-TM1 β-region on pore formation and dye uptake. A , FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor and reduced dye uptake though the N terminus mutants N16P, Y26L, S23N, and NRRL. Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow . Standard errors are only shown in one direction to not obscure the mean values. B , histogram summary showing the relative peak dye uptake for each receptor. Western blots show equivalent levels of surface receptor expression for P2X7 receptor WT and mutants. C , histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μ m BzATP addition made at 240 s). Data are mean ± S.E. **, p

    Techniques Used: Western Blot, Expressing

    Sucrose buffer enhances ethidium bromide dye uptake through the P2X7 receptor pore. A and B , representative FlexStation responses showing agonist-induced dye uptake through the rat ( A ) and human ( B ) P2X7 receptor when preloaded for 30 min with ethidium bromide (20 μ m ) under different buffer conditions (sucrose dye uptake buffer versus normal extracellular solution low divalent buffer). Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow. C , histogram summary showing the total RFU (relative fluorescence units) change under the different buffer loading conditions. D , histogram summary showing the -fold increase in dye uptake in sucrose dye uptake buffer compared with low divalent normal extracellular solution buffer. Data are mean ± S.E. ****, p
    Figure Legend Snippet: Sucrose buffer enhances ethidium bromide dye uptake through the P2X7 receptor pore. A and B , representative FlexStation responses showing agonist-induced dye uptake through the rat ( A ) and human ( B ) P2X7 receptor when preloaded for 30 min with ethidium bromide (20 μ m ) under different buffer conditions (sucrose dye uptake buffer versus normal extracellular solution low divalent buffer). Agonist addition (300 μ m BzATP) at 240 s is indicated by the arrow. C , histogram summary showing the total RFU (relative fluorescence units) change under the different buffer loading conditions. D , histogram summary showing the -fold increase in dye uptake in sucrose dye uptake buffer compared with low divalent normal extracellular solution buffer. Data are mean ± S.E. ****, p

    Techniques Used: Fluorescence

    Contribution of the pre-TM1 region to the time course of P2X1, P2X2, and P2X7 receptor currents. A , normalized traces demonstrating the effect on the time course of pre-TM1 β-region substitution of P2X1 into P2X7, P2X7 into P2X2, and P2X7 into P2X1 receptors, respectively. In each case, ATP was applied for the duration indicated by the gray bars (1 m m for P2X7 and P2X7–1Nβ and 100 μ m for P2X1 and P2X2 WT receptors and chimeras). B—D , histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse (EC 90 concentration of ATP applied for the duration as indicated on the adjacent traces). Western blots show the surface expression of the respective WT and chimeric receptors. For P2X2 and P2X7 receptors, the lanes are from the same blot and exposure, and the white line between them indicates that they have been reordered from that loaded on the gel. Data are mean ± S.E. ( n = 4–25). *, p
    Figure Legend Snippet: Contribution of the pre-TM1 region to the time course of P2X1, P2X2, and P2X7 receptor currents. A , normalized traces demonstrating the effect on the time course of pre-TM1 β-region substitution of P2X1 into P2X7, P2X7 into P2X2, and P2X7 into P2X1 receptors, respectively. In each case, ATP was applied for the duration indicated by the gray bars (1 m m for P2X7 and P2X7–1Nβ and 100 μ m for P2X1 and P2X2 WT receptors and chimeras). B—D , histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse (EC 90 concentration of ATP applied for the duration as indicated on the adjacent traces). Western blots show the surface expression of the respective WT and chimeric receptors. For P2X2 and P2X7 receptors, the lanes are from the same blot and exposure, and the white line between them indicates that they have been reordered from that loaded on the gel. Data are mean ± S.E. ( n = 4–25). *, p

    Techniques Used: Concentration Assay, Western Blot, Expressing

    4) Product Images from "The P2X7 Receptor Is Involved in Diabetic Neuropathic Pain Hypersensitivity Mediated by TRPV1 in the Rat Dorsal Root Ganglion"

    Article Title: The P2X7 Receptor Is Involved in Diabetic Neuropathic Pain Hypersensitivity Mediated by TRPV1 in the Rat Dorsal Root Ganglion

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2021.663649

    The coexpression of TRPV1 with NeuN and P2X 7 receptor with GFAP was detected by immunofluorescence staining. (A) Levels of TRPV1 with NeuN coexpression in neurons in the rat DRG ( n = 6 rats for each group). (B) The histogram shows the mean optical density of the coexpression of TRPV1 with NeuN. (C) Levels of P2X 7 with GFAP coexpression in SGCs in the rat DRG ( n = 6 rats for each group). (D) The histogram shows the mean optical density of the coexpression of the P2X 7 receptor with GFAP. In the DNP group, the coexpression of TRPV1 with NeuN in the DRG was increased compared with that in the control group, while the coexpression of TRPV1 with NeuN in the DNP + A438079 group was decreased compared with that in the DNP group. Furthermore, the coexpression of P2X 7 receptor with GFAP in the DRG was consistent with the coexpression trend described above. The green signal is fluorescein isothiocyanate (FITC)-labeled NeuN and FITC-labeled GFAP; the red signal is tetramethylrhodamine isothiocyanate (TRITC)-labeled TRPV1 and TRITC-labeled P2X 7 ; and the yellow signal is the combination of the green and red signals. The arrow indicates activated neuronal cells and SGCs in the DRG. The scale bar represents 50 μm. The data are presented as the mean ± SEM; compared with the control group, ** P
    Figure Legend Snippet: The coexpression of TRPV1 with NeuN and P2X 7 receptor with GFAP was detected by immunofluorescence staining. (A) Levels of TRPV1 with NeuN coexpression in neurons in the rat DRG ( n = 6 rats for each group). (B) The histogram shows the mean optical density of the coexpression of TRPV1 with NeuN. (C) Levels of P2X 7 with GFAP coexpression in SGCs in the rat DRG ( n = 6 rats for each group). (D) The histogram shows the mean optical density of the coexpression of the P2X 7 receptor with GFAP. In the DNP group, the coexpression of TRPV1 with NeuN in the DRG was increased compared with that in the control group, while the coexpression of TRPV1 with NeuN in the DNP + A438079 group was decreased compared with that in the DNP group. Furthermore, the coexpression of P2X 7 receptor with GFAP in the DRG was consistent with the coexpression trend described above. The green signal is fluorescein isothiocyanate (FITC)-labeled NeuN and FITC-labeled GFAP; the red signal is tetramethylrhodamine isothiocyanate (TRITC)-labeled TRPV1 and TRITC-labeled P2X 7 ; and the yellow signal is the combination of the green and red signals. The arrow indicates activated neuronal cells and SGCs in the DRG. The scale bar represents 50 μm. The data are presented as the mean ± SEM; compared with the control group, ** P

    Techniques Used: Immunofluorescence, Staining, Labeling

    The effect of A438079 on the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) of DNP rats. (A) Changes in the MWTs of rats from each group. (B) Changes in the TWLs of rats from each group. The MWTs and TWLs of rats from the DNP group were significantly lower than the values of rats from the control group (namely, the sensitivity was increased). At the end of the 8th week, the MWTs and TWLs of rats in the DNP group treated with an intrathecal injection of the P2X 7 receptor antagonist A438079 (DNP + A438079 group) were significantly higher than those of rats in the DNP group (i.e., decreased sensitivity). The data are presented as the mean ± SEM for the six animals in each group; compared with the control group, ** P
    Figure Legend Snippet: The effect of A438079 on the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) of DNP rats. (A) Changes in the MWTs of rats from each group. (B) Changes in the TWLs of rats from each group. The MWTs and TWLs of rats from the DNP group were significantly lower than the values of rats from the control group (namely, the sensitivity was increased). At the end of the 8th week, the MWTs and TWLs of rats in the DNP group treated with an intrathecal injection of the P2X 7 receptor antagonist A438079 (DNP + A438079 group) were significantly higher than those of rats in the DNP group (i.e., decreased sensitivity). The data are presented as the mean ± SEM for the six animals in each group; compared with the control group, ** P

    Techniques Used: Injection

    5) Product Images from "P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β"

    Article Title: P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20092298

    Paraffin sections from embedded PCLS after 300 mU/mL BLM exposure for 24 h ( A , B , E , F ) and 48 h ( C , D , G , H ). Immunoperoxidase demonstration of JAM-A in WT ( A – D ) and P2X7 −/− ( E – H ) mice. Note the preferable immunostaining of AECII in untreated WT (arrows in A , C ), a weak increase in P2X7 −/− mice ( E , G ), and the strongest immunostaining of the AECs in the BLM-treated WT mice ( B , D ). Arrowheads depict the alveolar lining of JAM-A immunoreactivity. Bar = 100 µm. Inset over ( A ) and ( E ): Analysis of mRNA content in paraffin sections of PCLS from WT and P2X7 −/− mice after 24 h. mRNA content of JAM-A was analyzed by quantitative real time RT-PCR using Hmbs and Rpl32 as housekeeping genes. Charts are represented as mean ± SEM (WT normalized to 1; n = 3; p -value 0.4550).
    Figure Legend Snippet: Paraffin sections from embedded PCLS after 300 mU/mL BLM exposure for 24 h ( A , B , E , F ) and 48 h ( C , D , G , H ). Immunoperoxidase demonstration of JAM-A in WT ( A – D ) and P2X7 −/− ( E – H ) mice. Note the preferable immunostaining of AECII in untreated WT (arrows in A , C ), a weak increase in P2X7 −/− mice ( E , G ), and the strongest immunostaining of the AECs in the BLM-treated WT mice ( B , D ). Arrowheads depict the alveolar lining of JAM-A immunoreactivity. Bar = 100 µm. Inset over ( A ) and ( E ): Analysis of mRNA content in paraffin sections of PCLS from WT and P2X7 −/− mice after 24 h. mRNA content of JAM-A was analyzed by quantitative real time RT-PCR using Hmbs and Rpl32 as housekeeping genes. Charts are represented as mean ± SEM (WT normalized to 1; n = 3; p -value 0.4550).

    Techniques Used: Mouse Assay, Immunostaining, Quantitative RT-PCR

    Frozen sections of mouse lung tissue. Immunofluorescence demonstration of JAM-A in WT and P2X7 −/− mice ( A – D ). Note the increase in immunoreactivity of JAM-A in P2X7 −/− mice ( B , D ). ( C , D ) Double immunofluorescence with the AECI marker T1α (TexasRed). Arrows show the linear pattern of JAM-A. Arrowheads depict examples of epithelial junctions. Bar = 100 µm. Corresponding mRNA ( E ) (Wildtype (WT) normalized to 1; n = 3; p -value 0.5737) and protein ( F ) ( n = 3, p -value 0.0357) levels in lung homogenates. * p
    Figure Legend Snippet: Frozen sections of mouse lung tissue. Immunofluorescence demonstration of JAM-A in WT and P2X7 −/− mice ( A – D ). Note the increase in immunoreactivity of JAM-A in P2X7 −/− mice ( B , D ). ( C , D ) Double immunofluorescence with the AECI marker T1α (TexasRed). Arrows show the linear pattern of JAM-A. Arrowheads depict examples of epithelial junctions. Bar = 100 µm. Corresponding mRNA ( E ) (Wildtype (WT) normalized to 1; n = 3; p -value 0.5737) and protein ( F ) ( n = 3, p -value 0.0357) levels in lung homogenates. * p

    Techniques Used: Immunofluorescence, Mouse Assay, Marker

    BLM treatment results in increased protein levels of P2X7R and JAM-A as well as in a reduced content of the inactive form of GSK-3β GSK-3β(Ser9). After inhibition of P2X7R by oxATP under BLM exposure, the effect on both proteins is further enhanced. Inactivating of the GSK-3β by LiCl under BLM exposure directly leads to a reduction of JAM-A. The influence of P2X7R on JAM-A is rather indirect.
    Figure Legend Snippet: BLM treatment results in increased protein levels of P2X7R and JAM-A as well as in a reduced content of the inactive form of GSK-3β GSK-3β(Ser9). After inhibition of P2X7R by oxATP under BLM exposure, the effect on both proteins is further enhanced. Inactivating of the GSK-3β by LiCl under BLM exposure directly leads to a reduction of JAM-A. The influence of P2X7R on JAM-A is rather indirect.

    Techniques Used: Inhibition

    Effects of P2X7R inhibition by 150 µM oxATP, which was added 2 h prior to 100 mU/mL BLM treatment. Equal protein amounts of cell lysates were used in SDS-PAGE and analyzed by Western blot. α-Tub served as the loading control. Untreated cells were used as the control and normalized to 100%. Representative blots from three independent experiments are shown. Charts are presented as the mean ± SEM ( n = 3) of P2X7R/ α-Tub, GSK-3β(Ser9)/ α-Tub and JAM-A/ α-Tub. P-values: P2X7R 0.0338; GSK-3β(Ser9) 0.002; and JAM-A 0.05. Immunofluorescence demonstration of JAM-A in untreated ( A ), BLM ( B ), or oxATP ( C ) treated E10 cells. Note the increased cell size after BLM exposure ( B ), which was ameliorated after oxATP ( D ). Representative images of multiple experiments ( n = 3) are shown. Bar = 20 µm. * p
    Figure Legend Snippet: Effects of P2X7R inhibition by 150 µM oxATP, which was added 2 h prior to 100 mU/mL BLM treatment. Equal protein amounts of cell lysates were used in SDS-PAGE and analyzed by Western blot. α-Tub served as the loading control. Untreated cells were used as the control and normalized to 100%. Representative blots from three independent experiments are shown. Charts are presented as the mean ± SEM ( n = 3) of P2X7R/ α-Tub, GSK-3β(Ser9)/ α-Tub and JAM-A/ α-Tub. P-values: P2X7R 0.0338; GSK-3β(Ser9) 0.002; and JAM-A 0.05. Immunofluorescence demonstration of JAM-A in untreated ( A ), BLM ( B ), or oxATP ( C ) treated E10 cells. Note the increased cell size after BLM exposure ( B ), which was ameliorated after oxATP ( D ). Representative images of multiple experiments ( n = 3) are shown. Bar = 20 µm. * p

    Techniques Used: Inhibition, SDS Page, Western Blot, Immunofluorescence

    Analysis of protein levels in alveolar epithelial cell line E10 after treatment with 150 µM BzATP. Cells were treated with BzATP for 24 h and 48 h. For SDS-PAGE, equal protein amounts of cell lysates were used and analyzed by Western blot with antibodies against P2X7R, JAM-A, and α-Tub. Untreated cells were used as the control and normalized to 100%. Protein levels were normalized to α-Tub and are shown as the mean ± SEM ( n = 3) in relation to the control. One representative blot is pictured. P-values: P2X7R 0.1667 and JAM-A 0.1048.
    Figure Legend Snippet: Analysis of protein levels in alveolar epithelial cell line E10 after treatment with 150 µM BzATP. Cells were treated with BzATP for 24 h and 48 h. For SDS-PAGE, equal protein amounts of cell lysates were used and analyzed by Western blot with antibodies against P2X7R, JAM-A, and α-Tub. Untreated cells were used as the control and normalized to 100%. Protein levels were normalized to α-Tub and are shown as the mean ± SEM ( n = 3) in relation to the control. One representative blot is pictured. P-values: P2X7R 0.1667 and JAM-A 0.1048.

    Techniques Used: SDS Page, Western Blot

    6) Product Images from "P2X7 Receptor Deficiency Ameliorates STZ-induced Cardiac Damage and Remodeling Through PKCβ and ERK"

    Article Title: P2X7 Receptor Deficiency Ameliorates STZ-induced Cardiac Damage and Remodeling Through PKCβ and ERK

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.692028

    Blockade of the P2X7 receptor improved STZ-induced cardiac apoptosis. Representative images (A) and quantification (C) of TUNEL staining in mouse myocardial tissues are shown (400× magnification). Levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured by western blot (B) . ( n = 3 per group). The ratio of the Bcl-2 protein to the Bax protein and the ratio of Caspase3/GAPDH proteins are shown (D,E) (data from three independent experiments were analyzed: * p
    Figure Legend Snippet: Blockade of the P2X7 receptor improved STZ-induced cardiac apoptosis. Representative images (A) and quantification (C) of TUNEL staining in mouse myocardial tissues are shown (400× magnification). Levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured by western blot (B) . ( n = 3 per group). The ratio of the Bcl-2 protein to the Bax protein and the ratio of Caspase3/GAPDH proteins are shown (D,E) (data from three independent experiments were analyzed: * p

    Techniques Used: TUNEL Assay, Staining, Western Blot

    Blockade of the P2X7 receptor attenuated STZ-induced cardiac remodeling. Representative images of hematoxylin-eosin staining (H E) of the myocardial tissues (400× magnification), ( A , upper panel). Myocardial fibrosis analysis was detected using Sirius Red staining ( A , middle panel), and representative images of Masson’s trichrome staining ( A , bottom panel) are shown (400× magnification). Bar graph showing the quantified interstitial fibrotic areas (%) in images of Sirius Red staining and Masson’s trichrome staining (C) . Levels of the MyHC, TGF-β, MMP-9, and COL-1 proteins in myocardial tissues were measured using western blotting (B) ( n = 3 per group). The mRNA expression of the hypertrophy markers MyHC and ANP (D,E) and fibrosis marker TGF-β (F) in the myocardial tissues is shown (data from three independent experiments were analyzed: * p
    Figure Legend Snippet: Blockade of the P2X7 receptor attenuated STZ-induced cardiac remodeling. Representative images of hematoxylin-eosin staining (H E) of the myocardial tissues (400× magnification), ( A , upper panel). Myocardial fibrosis analysis was detected using Sirius Red staining ( A , middle panel), and representative images of Masson’s trichrome staining ( A , bottom panel) are shown (400× magnification). Bar graph showing the quantified interstitial fibrotic areas (%) in images of Sirius Red staining and Masson’s trichrome staining (C) . Levels of the MyHC, TGF-β, MMP-9, and COL-1 proteins in myocardial tissues were measured using western blotting (B) ( n = 3 per group). The mRNA expression of the hypertrophy markers MyHC and ANP (D,E) and fibrosis marker TGF-β (F) in the myocardial tissues is shown (data from three independent experiments were analyzed: * p

    Techniques Used: Staining, Western Blot, Expressing, Aqueous Normal-phase Chromatography, Marker

    The P2X7 receptor was involved in STZ-induced cardiac damage. Representative images (A) of echocardiograms from each group of knockout mice. Representative images of hematoxylin-eosin staining (H E) of the myocardial tissues from knockout mice (400× magnification), ( B , upper panel). Myocardial fibrosis was analyzed using Masson’s trichrome staining, and representative images ( B , bottom panel) are shown (400× magnification). Quantification of the interstitial fibrotic areas (%) in images of Masson’s trichrome staining are shown in the bar graph (D) . The expression of the P2X7 receptor in myocardial tissues from knockout mice (C) . The protein levels of fibrosis markers in myocardial tissues were measured using western blotting (E,F) ( n = 3 animals per group). The levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured using western blotting (G) . The ratio of the Bcl-2 protein to the Bax protein and the ratio of Caspase3/GAPDH proteins are shown (H) . (Data from three independent experiments were analyzed: * p
    Figure Legend Snippet: The P2X7 receptor was involved in STZ-induced cardiac damage. Representative images (A) of echocardiograms from each group of knockout mice. Representative images of hematoxylin-eosin staining (H E) of the myocardial tissues from knockout mice (400× magnification), ( B , upper panel). Myocardial fibrosis was analyzed using Masson’s trichrome staining, and representative images ( B , bottom panel) are shown (400× magnification). Quantification of the interstitial fibrotic areas (%) in images of Masson’s trichrome staining are shown in the bar graph (D) . The expression of the P2X7 receptor in myocardial tissues from knockout mice (C) . The protein levels of fibrosis markers in myocardial tissues were measured using western blotting (E,F) ( n = 3 animals per group). The levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured using western blotting (G) . The ratio of the Bcl-2 protein to the Bax protein and the ratio of Caspase3/GAPDH proteins are shown (H) . (Data from three independent experiments were analyzed: * p

    Techniques Used: Knock-Out, Mouse Assay, Staining, Expressing, Western Blot

    P2X7 receptor inhibition prevented HG-induced phenotypic changes and apoptosis in H9c2 cells and rat primary cardiomyocytes. The levels of pro-fibrotic (MMP-9, TGF-β, and COL-1) and pro-hypertrophic proteins (MyHC) was examined using western blot analysis (A,B) . (E) The bar graph shows the corresponding PCR data for TGF-β, MYHC, and ANP. The same process and analysis were performed for rat primary cardiac myocytes. (WB: C,D , PCR: F ). Representative images of rhodamine staining in each group of H9c2 cells (G) . The levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured on H9c2 cells and primary cardiac myocytes using western blotting (H,I) . Flow cytometry analysis showing that A438079 reduced the apoptosis of HG-treated H9c2 cells (J) . (Data from three independent experiments were analyzed: * p
    Figure Legend Snippet: P2X7 receptor inhibition prevented HG-induced phenotypic changes and apoptosis in H9c2 cells and rat primary cardiomyocytes. The levels of pro-fibrotic (MMP-9, TGF-β, and COL-1) and pro-hypertrophic proteins (MyHC) was examined using western blot analysis (A,B) . (E) The bar graph shows the corresponding PCR data for TGF-β, MYHC, and ANP. The same process and analysis were performed for rat primary cardiac myocytes. (WB: C,D , PCR: F ). Representative images of rhodamine staining in each group of H9c2 cells (G) . The levels of the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were measured on H9c2 cells and primary cardiac myocytes using western blotting (H,I) . Flow cytometry analysis showing that A438079 reduced the apoptosis of HG-treated H9c2 cells (J) . (Data from three independent experiments were analyzed: * p

    Techniques Used: Inhibition, Western Blot, Polymerase Chain Reaction, Aqueous Normal-phase Chromatography, Staining, Flow Cytometry

    P2X7R regulated the activation of the PKCβ/ERK pathway in high glucose-induced cardiomyocytes. Total protein was extracted and p-PKC/PKC and p-ERK/ERK levels were analyzed using western blot analyses (A,B) . Levels of the P2X7R, p-PKC/PKC and p-ERK/ERK proteins were analyzed using western blotting (C) , and the quantitative statistics are presented in graphs (D–F) . The levels of pro-fibrotic, pro-apoptotic, and pro-hypertrophic proteins were determined using western blot analyses (G) . A schematic illustrating the role of P2X7R in diabetes/HG-induced injury in cardiomyocytes, the preventative effect of A438079 and the role of P2X7R knockout. (H) (Data from three independent experiments were analyzed: * p
    Figure Legend Snippet: P2X7R regulated the activation of the PKCβ/ERK pathway in high glucose-induced cardiomyocytes. Total protein was extracted and p-PKC/PKC and p-ERK/ERK levels were analyzed using western blot analyses (A,B) . Levels of the P2X7R, p-PKC/PKC and p-ERK/ERK proteins were analyzed using western blotting (C) , and the quantitative statistics are presented in graphs (D–F) . The levels of pro-fibrotic, pro-apoptotic, and pro-hypertrophic proteins were determined using western blot analyses (G) . A schematic illustrating the role of P2X7R in diabetes/HG-induced injury in cardiomyocytes, the preventative effect of A438079 and the role of P2X7R knockout. (H) (Data from three independent experiments were analyzed: * p

    Techniques Used: Activation Assay, Western Blot, Knock-Out

    P2X7 receptor expression was increased in the STZ-induced type 1 diabetes model and HG-treated cell model in vitro . Representative images (A) and quantification (B) of immunofluorescence staining for P2X7R in myocardial tissues (400× magnification). The expression of the P2X7R mRNA (C) and protein (D) with the corresponding statistics (E) (data from three independent experiments were analyzed: * p
    Figure Legend Snippet: P2X7 receptor expression was increased in the STZ-induced type 1 diabetes model and HG-treated cell model in vitro . Representative images (A) and quantification (B) of immunofluorescence staining for P2X7R in myocardial tissues (400× magnification). The expression of the P2X7R mRNA (C) and protein (D) with the corresponding statistics (E) (data from three independent experiments were analyzed: * p

    Techniques Used: Expressing, In Vitro, Immunofluorescence, Staining

    7) Product Images from "P2x7 receptors control demyelination and inflammation in the cuprizone model"

    Article Title: P2x7 receptors control demyelination and inflammation in the cuprizone model

    Journal: Brain, Behavior, & Immunity - Health

    doi: 10.1016/j.bbih.2020.100062

    P2x7 receptors are not master regulators of demyelination-associated microglia phenotype. Wild-type and P2x7 knockout mice were treated with cuprizone for 6 weeks and microglial cells purified from the forebrain using flow cytometry. RT-qPCR analysis of inflammasome activationmarkers ( A ) and M1/M2 phenotype genes ( C ) in cell sorted from control and cuprizone-treated mice showed selective up-regulation of several pro-inflammatory genes in demyelination-associated microglia. Data represent relative fold change compared with microglial cells purified from control mice normalized to B2m and Ppia ( n ​= ​3-6 mice per group). ( B , D ) Relative expression of indicate genes in microglia sorted from cuprizone-treated P2x7 knockout mice ( n ​= ​3-6 mice per group; fold change compared with expression levels in cells purified from cuprizone-treated wild-type mice; normalized to B2m and Ppia ). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: P2x7 receptors are not master regulators of demyelination-associated microglia phenotype. Wild-type and P2x7 knockout mice were treated with cuprizone for 6 weeks and microglial cells purified from the forebrain using flow cytometry. RT-qPCR analysis of inflammasome activationmarkers ( A ) and M1/M2 phenotype genes ( C ) in cell sorted from control and cuprizone-treated mice showed selective up-regulation of several pro-inflammatory genes in demyelination-associated microglia. Data represent relative fold change compared with microglial cells purified from control mice normalized to B2m and Ppia ( n ​= ​3-6 mice per group). ( B , D ) Relative expression of indicate genes in microglia sorted from cuprizone-treated P2x7 knockout mice ( n ​= ​3-6 mice per group; fold change compared with expression levels in cells purified from cuprizone-treated wild-type mice; normalized to B2m and Ppia ). CPZ, cuprizone. ∗ p ​

    Techniques Used: Knock-Out, Mouse Assay, Purification, Flow Cytometry, Quantitative RT-PCR, Expressing

    Pharmacological blockade of P2x7 receptors does not improve spontaneous remyelination in the cuprizone model. Mice received cuprizone in the diet for 6 weeks and BBG (50 ​mg/Kg), JNJ-47965567 (JNJ; 30 ​mg/Kg) or vehicle solutions during a 2 week remyelination phase. ( A , B ) LFB and MBP immunostaining revealed extensive demyelination at 6 weeks of cuprizone administration and partial recovery 2 weeks after toxin withdrawal. Scoring of LFB and MBP staining showed no differences between mice treated with BBG ( A ), JNJ-47965567 ( B ) or vehicle during the recovery phase ( n ​= ​4-6 mice per group). ∗∗∗ p ​
    Figure Legend Snippet: Pharmacological blockade of P2x7 receptors does not improve spontaneous remyelination in the cuprizone model. Mice received cuprizone in the diet for 6 weeks and BBG (50 ​mg/Kg), JNJ-47965567 (JNJ; 30 ​mg/Kg) or vehicle solutions during a 2 week remyelination phase. ( A , B ) LFB and MBP immunostaining revealed extensive demyelination at 6 weeks of cuprizone administration and partial recovery 2 weeks after toxin withdrawal. Scoring of LFB and MBP staining showed no differences between mice treated with BBG ( A ), JNJ-47965567 ( B ) or vehicle during the recovery phase ( n ​= ​4-6 mice per group). ∗∗∗ p ​

    Techniques Used: Mouse Assay, Immunostaining, Staining

    Effect of P2x7 receptor antagonists on glial cells and inflammatory responses during remyelination. ( A ) Quantification of CD11b + and Iba1 + cells and GFAP immunoreactivity following treatment with P2x7 receptor antagonists. Treatment with BBG or JNJ-47965567 during recovery from cuprizone intoxication did not modulate the presence of microglia/macrophages or astrocytes in remyelinating corpus callosum ( n ​= ​5-6 mice per group). ( B , C ) Expression of inflammasome- and polarization-related molecules assessed by RT-qPCR in brain tissue from mice treated with BBG ( B ) or JNJ-47965567 ( C ). Data represent fold change relative to vehicle-treated mice normalized to Gapdh , Hprt1 and Ppia ( n ​= ​5-6 mice per group). ∗ p ​
    Figure Legend Snippet: Effect of P2x7 receptor antagonists on glial cells and inflammatory responses during remyelination. ( A ) Quantification of CD11b + and Iba1 + cells and GFAP immunoreactivity following treatment with P2x7 receptor antagonists. Treatment with BBG or JNJ-47965567 during recovery from cuprizone intoxication did not modulate the presence of microglia/macrophages or astrocytes in remyelinating corpus callosum ( n ​= ​5-6 mice per group). ( B , C ) Expression of inflammasome- and polarization-related molecules assessed by RT-qPCR in brain tissue from mice treated with BBG ( B ) or JNJ-47965567 ( C ). Data represent fold change relative to vehicle-treated mice normalized to Gapdh , Hprt1 and Ppia ( n ​= ​5-6 mice per group). ∗ p ​

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR

    Up-regulated P2x7 receptor expression and signalling during cuprizone intoxication. ( A ) RT-qPCR analyses showed significant increases in P2rx7 expression during the time-course of cuprizone administration ( n ​= ​7 mice per group; normalized to Gapdh and Hprt1 . ( B ) Up-regulated P2x7 receptor levels were detected following 6 weeks of cuprizone feeding ( n ​= ​5 mice per group). At each treatment point, brain slices from cuprizone-treated mice were compared with tissue from mice fed a control diet. ( C ) Representative images of P2x7 receptor immunostaining in the corpus callosum of mice treated with a cuprizone containing diet for 6 weeks and untreated controls. Quantification of immunolabelled tissue sections revealed significant up-regulation of P2x7 receptors in demyelinated corpus callosum ( n ​= ​4 mice per group). ( D ) Increased expression of inflammasome-related molecules following 6 weeks of cuprizone intoxication. Transcript levels of indicated genes were assessed by RT-qPCR. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: Up-regulated P2x7 receptor expression and signalling during cuprizone intoxication. ( A ) RT-qPCR analyses showed significant increases in P2rx7 expression during the time-course of cuprizone administration ( n ​= ​7 mice per group; normalized to Gapdh and Hprt1 . ( B ) Up-regulated P2x7 receptor levels were detected following 6 weeks of cuprizone feeding ( n ​= ​5 mice per group). At each treatment point, brain slices from cuprizone-treated mice were compared with tissue from mice fed a control diet. ( C ) Representative images of P2x7 receptor immunostaining in the corpus callosum of mice treated with a cuprizone containing diet for 6 weeks and untreated controls. Quantification of immunolabelled tissue sections revealed significant up-regulation of P2x7 receptors in demyelinated corpus callosum ( n ​= ​4 mice per group). ( D ) Increased expression of inflammasome-related molecules following 6 weeks of cuprizone intoxication. Transcript levels of indicated genes were assessed by RT-qPCR. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). CPZ, cuprizone. ∗ p ​

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Immunostaining

    P2x7 receptor deficient mice are resistant to cuprizone-induced demyelination. ( A ) P2x7 +/+ and P2x7 -/- mice were fed 0.3% cuprizone in the diet for 6 weeks. Analysis of tissue sections stained for LFB and MBP showed attenuated myelin loss in P2x7 receptor deficient mice ( n ​= ​3-5 mice). Representative images depict the loss of MBP immunolabelling after cuprizone administration in P2x7 +/+ and P2x7 -/- mice. ( B-D ) The corpus callosum was immunostained for CD11b, GFAP, NG2, CD3, MAC387 and myeloperoxidase (MPO) as markers of microglia/macrophages, astrocytes, OPCs, T cells, recently infiltrating monocytes/macrophages and neutrophils, respectively. Quantification and representative images show a reduced accumulation of astrocytes and microglia in P2x7 -/- mice at 6 weeks of cuprizone diet ( B ), but no differences regarding the recruitment of OPCs ( C ) or peripheral immune cells ( D ) to demyelinated tissue ( n ​= ​4-5 mice). ( E ) Transcript levels of inflammasome-associated genes were assessed by RT-qPCR in brain tissue from P2x7 +/+ and P2x7 -/- mice at 6 weeks of cuprizone intoxication. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). Scale bars: 200 ​μm ( A ) and 50 ​μm ( B ). CPZ, cuprizone; N.D., not detected. ∗ p ​
    Figure Legend Snippet: P2x7 receptor deficient mice are resistant to cuprizone-induced demyelination. ( A ) P2x7 +/+ and P2x7 -/- mice were fed 0.3% cuprizone in the diet for 6 weeks. Analysis of tissue sections stained for LFB and MBP showed attenuated myelin loss in P2x7 receptor deficient mice ( n ​= ​3-5 mice). Representative images depict the loss of MBP immunolabelling after cuprizone administration in P2x7 +/+ and P2x7 -/- mice. ( B-D ) The corpus callosum was immunostained for CD11b, GFAP, NG2, CD3, MAC387 and myeloperoxidase (MPO) as markers of microglia/macrophages, astrocytes, OPCs, T cells, recently infiltrating monocytes/macrophages and neutrophils, respectively. Quantification and representative images show a reduced accumulation of astrocytes and microglia in P2x7 -/- mice at 6 weeks of cuprizone diet ( B ), but no differences regarding the recruitment of OPCs ( C ) or peripheral immune cells ( D ) to demyelinated tissue ( n ​= ​4-5 mice). ( E ) Transcript levels of inflammasome-associated genes were assessed by RT-qPCR in brain tissue from P2x7 +/+ and P2x7 -/- mice at 6 weeks of cuprizone intoxication. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). Scale bars: 200 ​μm ( A ) and 50 ​μm ( B ). CPZ, cuprizone; N.D., not detected. ∗ p ​

    Techniques Used: Mouse Assay, Staining, Quantitative RT-PCR

    P2x7 receptors promote the accumulation of pro-inflammatory microglia during cuprizone-induced demyelination. ( A ) RT-qPCR analysis of M1 and M2 signature genes in brain tissue from mice treated with a cuprizone-containing diet for 6 weeks. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). ( B ) P2x7 knockout mice displayed down-regulated induction of M1 phenotype markers at 6 weeks of cuprizone intoxication. Results are expressed as relative fold change relative to controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​6 mice per group). ( C-D ) Quantification and representative images depicting iNOS and Arg-1 colocalization with CD11b in mice treated with cuprizone for 6 weeks. P2x7 -/- mice exhibited a significant reduction in the number of CD11b + cells expressing iNOS in demyelinated corpus callosum. Scale bars: 20 ​μm. CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: P2x7 receptors promote the accumulation of pro-inflammatory microglia during cuprizone-induced demyelination. ( A ) RT-qPCR analysis of M1 and M2 signature genes in brain tissue from mice treated with a cuprizone-containing diet for 6 weeks. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). ( B ) P2x7 knockout mice displayed down-regulated induction of M1 phenotype markers at 6 weeks of cuprizone intoxication. Results are expressed as relative fold change relative to controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​6 mice per group). ( C-D ) Quantification and representative images depicting iNOS and Arg-1 colocalization with CD11b in mice treated with cuprizone for 6 weeks. P2x7 -/- mice exhibited a significant reduction in the number of CD11b + cells expressing iNOS in demyelinated corpus callosum. Scale bars: 20 ​μm. CPZ, cuprizone. ∗ p ​

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Knock-Out, Expressing

    Cell-specific analysis of P2x7 receptor expression in cuprizone-treated mice. Confocal images corresponding to double immunostaining of P2x7 receptors and the microglia/macrophage marker CD11b ( A ) and the astrocytic marker GFAP ( B ) in the corpus callosum of mice fed a cuprizone containing diet for 6 weeks. ( C-D ) Analysis of confocal fluorescent images indicated higher colocalization of P2x7 receptor immunopositive puncta with CD11b + microglia than with GFAP + astrocytic profiles, both in control and in cuprizone-treated mice ( n ​= ​4 mice per group). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: Cell-specific analysis of P2x7 receptor expression in cuprizone-treated mice. Confocal images corresponding to double immunostaining of P2x7 receptors and the microglia/macrophage marker CD11b ( A ) and the astrocytic marker GFAP ( B ) in the corpus callosum of mice fed a cuprizone containing diet for 6 weeks. ( C-D ) Analysis of confocal fluorescent images indicated higher colocalization of P2x7 receptor immunopositive puncta with CD11b + microglia than with GFAP + astrocytic profiles, both in control and in cuprizone-treated mice ( n ​= ​4 mice per group). CPZ, cuprizone. ∗ p ​

    Techniques Used: Expressing, Mouse Assay, Double Immunostaining, Marker

    8) Product Images from "Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma"

    Article Title: Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-021-02366-x

    Pro-inflammatory factor release and dynamic changes of M1-like and M2-like genotypes of activated microglia. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in primary cultured microglia which were treated with 100 μM BzATP for different times. b , c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained at different times of treatment is shown in the bar charts ( b for TSPO; c for P2X7R). d Immunofluorescent labeling showing the changes in morphology and TSPO expression in primary cultured microglia after the cells were treated with 100 μM BzATP for different periods of time. Scale bar: 20 μm for all images. e, f Cumulative data summarizing the changes in mRNA levels of IL-6 ( e ) and TNF-α ( f ) in cultured microglia extracts obtained in Ctr and those with BzATP treatment for different periods of time. g, h Bar chart showing the average extracellular IL-6 ( g ) and TNF-α ( h ) concentrations in cultured microglia in Ctr and groups of BzATP treatment for different periods of time. i Double immunofluorescent labeling showing the dynamic changes in expression of CD86 and CD206 in primary cultured microglia after the cells were treated with 100 μM BzATP for different periods of time. Scale bar: 20 μm for all images. * P
    Figure Legend Snippet: Pro-inflammatory factor release and dynamic changes of M1-like and M2-like genotypes of activated microglia. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in primary cultured microglia which were treated with 100 μM BzATP for different times. b , c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained at different times of treatment is shown in the bar charts ( b for TSPO; c for P2X7R). d Immunofluorescent labeling showing the changes in morphology and TSPO expression in primary cultured microglia after the cells were treated with 100 μM BzATP for different periods of time. Scale bar: 20 μm for all images. e, f Cumulative data summarizing the changes in mRNA levels of IL-6 ( e ) and TNF-α ( f ) in cultured microglia extracts obtained in Ctr and those with BzATP treatment for different periods of time. g, h Bar chart showing the average extracellular IL-6 ( g ) and TNF-α ( h ) concentrations in cultured microglia in Ctr and groups of BzATP treatment for different periods of time. i Double immunofluorescent labeling showing the dynamic changes in expression of CD86 and CD206 in primary cultured microglia after the cells were treated with 100 μM BzATP for different periods of time. Scale bar: 20 μm for all images. * P

    Techniques Used: Western Blot, Expressing, Cell Culture, Labeling

    P2X7R/Ca 2+ /NFAT/NF-kB pathway mediates microglial inflammatory response. a BzATP-induced changes in intracellular Ca 2+ concentrations ([Ca 2+ ] i ), indicated by F 340/380 , in microglial cells acutely isolated from control (Ctr) and COH retinas of the MacGreen mice at different post-operational times. b – e Bar charts summarizing the changes of peak value of F 340/380 ( b ), delay time ( c ), time to peak ( d ), and recovery ( e ) in microglial cells of Ctr and cells of COH retinas at different post-operational times. n = 10 for each group. * P
    Figure Legend Snippet: P2X7R/Ca 2+ /NFAT/NF-kB pathway mediates microglial inflammatory response. a BzATP-induced changes in intracellular Ca 2+ concentrations ([Ca 2+ ] i ), indicated by F 340/380 , in microglial cells acutely isolated from control (Ctr) and COH retinas of the MacGreen mice at different post-operational times. b – e Bar charts summarizing the changes of peak value of F 340/380 ( b ), delay time ( c ), time to peak ( d ), and recovery ( e ) in microglial cells of Ctr and cells of COH retinas at different post-operational times. n = 10 for each group. * P

    Techniques Used: Isolation, Mouse Assay

    ATP released from activated Müller cells induces microglia activation through P2X7R. a–c Representative immunoblots showing the changes of GFAP and TSPO expression in retinas with intravitreal injections of DHPG (100 μM, 2 μL) ( a ), DHPG along with Gap26 (200 μM, 2 μL) ( b ), and DHPG along with BBG (10 μM, 2 μL) ( c ) at different times after the injections. d , e A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( d for GFAP; e for TSPO). f Representative immunoblots showing the changes of GFAP and TSPO expression at different post-operational times in normal and COH retinas with intravitreal injections of saline (2 μL)/20 μM MPEP (2 μL). Saline or MPEP injections were made two days before COH operation. g , h A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( g for TSPO; h for GFAP). i Representative immunoblots showing the changes in TSPO and GFAP protein expression in COH retinas with or without the intravitreal injection of BBG. 2 μL BBG (10 μM) or normal saline was intravitreally injected 2 day before the COH operation. j , k A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( j for TSPO; k for GFAP). l , m Western blot results showing the changes in TSPO protein expression in COH retinas with or without the intravitreal injection of 5-BDBD, a P2X4R antagonist. All the data are normalized to control. In all experiments, n = 6 for each group. * P
    Figure Legend Snippet: ATP released from activated Müller cells induces microglia activation through P2X7R. a–c Representative immunoblots showing the changes of GFAP and TSPO expression in retinas with intravitreal injections of DHPG (100 μM, 2 μL) ( a ), DHPG along with Gap26 (200 μM, 2 μL) ( b ), and DHPG along with BBG (10 μM, 2 μL) ( c ) at different times after the injections. d , e A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( d for GFAP; e for TSPO). f Representative immunoblots showing the changes of GFAP and TSPO expression at different post-operational times in normal and COH retinas with intravitreal injections of saline (2 μL)/20 μM MPEP (2 μL). Saline or MPEP injections were made two days before COH operation. g , h A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( g for TSPO; h for GFAP). i Representative immunoblots showing the changes in TSPO and GFAP protein expression in COH retinas with or without the intravitreal injection of BBG. 2 μL BBG (10 μM) or normal saline was intravitreally injected 2 day before the COH operation. j , k A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts ( j for TSPO; k for GFAP). l , m Western blot results showing the changes in TSPO protein expression in COH retinas with or without the intravitreal injection of 5-BDBD, a P2X4R antagonist. All the data are normalized to control. In all experiments, n = 6 for each group. * P

    Techniques Used: Activation Assay, Western Blot, Expressing, Injection

    Activated Müller cells cause microglia activation via ATP/P2X7R pathway. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the P2X7R blocker BBG. Müller cells were pre-activated by incubating with DHPG for 72 h. BBG (10 μM) was added to culture medium of microglia 2 h before the activated Müller cells were added to the Transwell system. b , c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts ( b for TSPO; c for P2X7R). d Representative western blotting results showing the changes in protein levels of TSPO and P2X7R in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the Cx43 blocker Gap62. Gap26 (200 μM) was added to culture medium of Müller cells. e , f A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts ( e for TSPO; f for P2X7R). All the data are normalized to empty transwell group. * P
    Figure Legend Snippet: Activated Müller cells cause microglia activation via ATP/P2X7R pathway. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the P2X7R blocker BBG. Müller cells were pre-activated by incubating with DHPG for 72 h. BBG (10 μM) was added to culture medium of microglia 2 h before the activated Müller cells were added to the Transwell system. b , c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts ( b for TSPO; c for P2X7R). d Representative western blotting results showing the changes in protein levels of TSPO and P2X7R in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the Cx43 blocker Gap62. Gap26 (200 μM) was added to culture medium of Müller cells. e , f A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts ( e for TSPO; f for P2X7R). All the data are normalized to empty transwell group. * P

    Techniques Used: Activation Assay, Western Blot, Expressing, Cell Culture

    P2X7R-mediated changes of microglia in COH retinas. a Immunofluorescence staining showing changes in morphology and migration of the Iba1-labeled microglia, and in TSPO expression in retinal vertical slices taken from sham-operated P2X7R −/− mouse (Control, Ctr), and P2X7R −/− mice obtained at different post-operational times (G4d, G1w, G3w, and G5w). Scale bar: 10 μm for all images. b Representative western blotting results showing the changes in protein levels of P2X7R, P2X4R, TSPO, and GFAP in Ctr and COH retinas obtained from WT and P2X7R −/− mice at different post-operational times. c – f Bar charts comparing the average relative densities of immunoreactive bands of P2X7R ( c ), TSPO ( d ), GFAP ( e ) and P2X4R ( f ) expression obtained at corresponding post-operational times in COH retinas of wild type and P2X7R −/− mice. All the data are normalized to the corresponding controls. n = 6 for each group. * P
    Figure Legend Snippet: P2X7R-mediated changes of microglia in COH retinas. a Immunofluorescence staining showing changes in morphology and migration of the Iba1-labeled microglia, and in TSPO expression in retinal vertical slices taken from sham-operated P2X7R −/− mouse (Control, Ctr), and P2X7R −/− mice obtained at different post-operational times (G4d, G1w, G3w, and G5w). Scale bar: 10 μm for all images. b Representative western blotting results showing the changes in protein levels of P2X7R, P2X4R, TSPO, and GFAP in Ctr and COH retinas obtained from WT and P2X7R −/− mice at different post-operational times. c – f Bar charts comparing the average relative densities of immunoreactive bands of P2X7R ( c ), TSPO ( d ), GFAP ( e ) and P2X4R ( f ) expression obtained at corresponding post-operational times in COH retinas of wild type and P2X7R −/− mice. All the data are normalized to the corresponding controls. n = 6 for each group. * P

    Techniques Used: Immunofluorescence, Staining, Migration, Labeling, Expressing, Mouse Assay, Western Blot

    9) Product Images from "Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium"

    Article Title: Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium

    Journal: Analytical Cellular Pathology (Amsterdam)

    doi: 10.1155/2021/4793338

    Association of pannexin1 and P2X7 in unwounded corneas and 2 hrs after injury in DiO and B6 control corneal epithelium. Proximity ligation assays were performed using antibodies to P2X7 and pannexin1. (a) Representative enface and orthogonal (ortho) images of B6 control and DiO corneas are shown. The green color displays puncta of association. Corneal epithelium was counter stained with rhodamine phalloidin (asterisk marks wound area). The numbers and letters mark the individual cells that were analyzed using CellProfiler near the wound and back from the wound. The boxes indicate the representative individual cells that are shown in (b) after PLA analysis with CellProfiler. Bar equals 20 microns (PLA enface and actin) or 15 microns (PLA ortho). (b) Analysis of overlapping puncta for each condition (unwounded, leading edge, back from leading edge) was performed and a box plot drawn using R. The mean puncta for each region are placed above the box. Mean ± SEM are plotted, and two-way ANOVA with Tukey's multiple comparison of means was performed. p
    Figure Legend Snippet: Association of pannexin1 and P2X7 in unwounded corneas and 2 hrs after injury in DiO and B6 control corneal epithelium. Proximity ligation assays were performed using antibodies to P2X7 and pannexin1. (a) Representative enface and orthogonal (ortho) images of B6 control and DiO corneas are shown. The green color displays puncta of association. Corneal epithelium was counter stained with rhodamine phalloidin (asterisk marks wound area). The numbers and letters mark the individual cells that were analyzed using CellProfiler near the wound and back from the wound. The boxes indicate the representative individual cells that are shown in (b) after PLA analysis with CellProfiler. Bar equals 20 microns (PLA enface and actin) or 15 microns (PLA ortho). (b) Analysis of overlapping puncta for each condition (unwounded, leading edge, back from leading edge) was performed and a box plot drawn using R. The mean puncta for each region are placed above the box. Mean ± SEM are plotted, and two-way ANOVA with Tukey's multiple comparison of means was performed. p

    Techniques Used: Ligation, Staining, Proximity Ligation Assay

    10) Product Images from "P2X7 Receptor and Heart Function in a Mouse Model of Systemic Inflammation Due to High Fat Diet"

    Article Title: P2X7 Receptor and Heart Function in a Mouse Model of Systemic Inflammation Due to High Fat Diet

    Journal: Journal of Inflammation Research

    doi: 10.2147/JIR.S356038

    Heart gene expression profile of key regulators of oxidative stress modulation, endothelial function and chemotactic processes observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-HFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), Nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ), Nitric oxide synthase 3, endothelial (NOS-3, D ), Adhesion G protein-coupled receptor E1 (ADGRE1, E ), Intercellular adhesion molecule 1 (ICAM-1, F ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Heart gene expression profile of key regulators of oxidative stress modulation, endothelial function and chemotactic processes observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-HFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), Nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ), Nitric oxide synthase 3, endothelial (NOS-3, D ), Adhesion G protein-coupled receptor E1 (ADGRE1, E ), Intercellular adhesion molecule 1 (ICAM-1, F ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Expressing, Mouse Assay, Knock-Out

    Heart miRNA expression observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). miR-27a ( A ), miR-27b ( B ), miR-214 ( C ), miR-126 ( D ), miR-21 ( E ) are shown. Relative miRNA expression was calculated by the 2 −ΔΔCt method, using the reference miR-16-5p and miR-191-5p for normalization. Results are reported as arbitrary units (A.U.). Data are presented as mean values ±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Heart miRNA expression observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). miR-27a ( A ), miR-27b ( B ), miR-214 ( C ), miR-126 ( D ), miR-21 ( E ) are shown. Relative miRNA expression was calculated by the 2 −ΔΔCt method, using the reference miR-16-5p and miR-191-5p for normalization. Results are reported as arbitrary units (A.U.). Data are presented as mean values ±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Expressing, Mouse Assay, Knock-Out

    Weight ( A ) and plasma glucose (GLUC, B ), triglycerides (TRG, C ), cholesterol (CHL, D ), and aspartate aminotransferase (AST, E ) levels in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Weight ( A ) and plasma glucose (GLUC, B ), triglycerides (TRG, C ), cholesterol (CHL, D ), and aspartate aminotransferase (AST, E ) levels in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: AST Assay, Mouse Assay, Knock-Out

    Representative Western blots of protein expression of Interleukin-1β (IL-1β, A ), Interleukin-6 (IL-6, B ) and p38/mitogen-activated protein kinase and its phosphorylated form (p38MAPK and p-p38MAPK, C ) in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Band intensities were normalized based on reference protein GAPDH or Ponceau S and results are expressed as arbitrary units (A.U.). Arrows indicate different IL-1β isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Representative Western blots of protein expression of Interleukin-1β (IL-1β, A ), Interleukin-6 (IL-6, B ) and p38/mitogen-activated protein kinase and its phosphorylated form (p38MAPK and p-p38MAPK, C ) in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Band intensities were normalized based on reference protein GAPDH or Ponceau S and results are expressed as arbitrary units (A.U.). Arrows indicate different IL-1β isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Western Blot, Expressing, Mouse Assay, Knock-Out

    Heart gene expression of NLRP3-inflammasome complex and pro-inflammatory cytokines in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). NLR family, pyrin domain containing 3 (NLRP3, A ), Caspase-1 (CASP-1, B ), Nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (NFkB, C ), Interleukin-1β (IL-1β, D ), Interleukin-6 (IL-6, E ) and Interleukin-18 (IL-18, F ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Heart gene expression of NLRP3-inflammasome complex and pro-inflammatory cytokines in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). NLR family, pyrin domain containing 3 (NLRP3, A ), Caspase-1 (CASP-1, B ), Nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (NFkB, C ), Interleukin-1β (IL-1β, D ), Interleukin-6 (IL-6, E ) and Interleukin-18 (IL-18, F ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Expressing, Mouse Assay, Knock-Out

    Heart gene expression profile of biomarkers associated to pro-fibrotic process observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Transforming growth factor β1 (TGFβ, A ), Collagen, type I, alpha 2 (COL1A2, B ), Collagen, type III, alpha 1 (COL3A1, C ), Lysyl oxidase-like 2 (LOXL2, D ), Matrix metallopeptidase 9 (MMP-9, E ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Heart gene expression profile of biomarkers associated to pro-fibrotic process observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Transforming growth factor β1 (TGFβ, A ), Collagen, type I, alpha 2 (COL1A2, B ), Collagen, type III, alpha 1 (COL3A1, C ), Lysyl oxidase-like 2 (LOXL2, D ), Matrix metallopeptidase 9 (MMP-9, E ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Expressing, Mouse Assay, Knock-Out

    Cardiac performance of wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Ratio of the early to late ventricular filling velocities (E/A ratio, A ); left ventricular mass (LVmass, B ); cardiac output (CO, C ); ejection fraction (EF, D ); fractional area change (FAC, E ); fractional shortening (FS, F ); stroke volume (SV, G ) are shown. Measures were obtained by high frequency-ultrasound (UHFUS) examination, as described in Materials and Methods. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Cardiac performance of wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Ratio of the early to late ventricular filling velocities (E/A ratio, A ); left ventricular mass (LVmass, B ); cardiac output (CO, C ); ejection fraction (EF, D ); fractional area change (FAC, E ); fractional shortening (FS, F ); stroke volume (SV, G ) are shown. Measures were obtained by high frequency-ultrasound (UHFUS) examination, as described in Materials and Methods. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Mouse Assay, Knock-Out

    Heart protein expression of key regulators of oxidative stress modulation, endothelial function and chemotactic processes whose genes were differentially expressed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ) are shown. Band intensities were normalized based on reference protein GAPDH and results are expressed as arbitrary units (A.U.). Arrows indicate different MCP-1 isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: Heart protein expression of key regulators of oxidative stress modulation, endothelial function and chemotactic processes whose genes were differentially expressed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ) are shown. Band intensities were normalized based on reference protein GAPDH and results are expressed as arbitrary units (A.U.). Arrows indicate different MCP-1 isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Expressing, Mouse Assay, Knock-Out

    ( A ) Representative images of Picrosirius staining for quantification of heart fibrosis in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 purinergic receptor (P2X7R) receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). A positive control (db/db mice treated with HFD, CTL+) is also shown. Magnification: x40. Scale bar: 20μm. A staining quantification in the four groups of animals is shown. The threshold analysis was performed as described in Materials and Methods section. ( B ) Representative Western blot of TGFβ heart protein expression in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7R knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Results are expressed as arbitrary units (A.U.). Arrows indicate different TGFβ isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p
    Figure Legend Snippet: ( A ) Representative images of Picrosirius staining for quantification of heart fibrosis in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 purinergic receptor (P2X7R) receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). A positive control (db/db mice treated with HFD, CTL+) is also shown. Magnification: x40. Scale bar: 20μm. A staining quantification in the four groups of animals is shown. The threshold analysis was performed as described in Materials and Methods section. ( B ) Representative Western blot of TGFβ heart protein expression in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7R knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Results are expressed as arbitrary units (A.U.). Arrows indicate different TGFβ isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p

    Techniques Used: Staining, Mouse Assay, Knock-Out, Positive Control, Western Blot, Expressing

    11) Product Images from "P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine"

    Article Title: P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine

    Journal: The Journal of Headache and Pain

    doi: 10.1186/s10194-022-01442-8

    Schematic diagram for the mechanisms of P2X7R in the regulation of inflammasome priming, activation and programmed cell death pathways in the brain following repeated dural IS stimulation. The expression of P2X7R is upregulated after repeated dural IS stimulation. P2X7R can activate the assembly of NLRP3 inflammasome, a multi-protein complex including NLRP3, adaptor (i.e., ASC) and effector proteins (i.e., total caspase-1). The formation of an inflammasome complex activates and converts total caspase-1 into active cleaved caspase-1. There are three main groups of substrates that are targeted by cleaved caspase-1. Firstly, cleaved caspase-1 can cleave both precursor IL-1β and IL-18 into active proinflammatory cytokines, mature IL-1β and IL-18. Secondly, cleaved ca spase-1 can cleave GSDMD-FL into GSDMD-NT that self-oligomerize onto the plasma membrane to form a pore to facilitate the influx of water molecules to induce a lytic form of cell death known as pyroptosis. Thirdly, cleaved caspase-1 can cleave and activate total caspase-3 into cleave caspase-3 to induce apoptosis. Abbreviations: GSDMD-FL, full length Gasdermin D; GSDMD-NT, N-terminal Gasdermin D
    Figure Legend Snippet: Schematic diagram for the mechanisms of P2X7R in the regulation of inflammasome priming, activation and programmed cell death pathways in the brain following repeated dural IS stimulation. The expression of P2X7R is upregulated after repeated dural IS stimulation. P2X7R can activate the assembly of NLRP3 inflammasome, a multi-protein complex including NLRP3, adaptor (i.e., ASC) and effector proteins (i.e., total caspase-1). The formation of an inflammasome complex activates and converts total caspase-1 into active cleaved caspase-1. There are three main groups of substrates that are targeted by cleaved caspase-1. Firstly, cleaved caspase-1 can cleave both precursor IL-1β and IL-18 into active proinflammatory cytokines, mature IL-1β and IL-18. Secondly, cleaved ca spase-1 can cleave GSDMD-FL into GSDMD-NT that self-oligomerize onto the plasma membrane to form a pore to facilitate the influx of water molecules to induce a lytic form of cell death known as pyroptosis. Thirdly, cleaved caspase-1 can cleave and activate total caspase-3 into cleave caspase-3 to induce apoptosis. Abbreviations: GSDMD-FL, full length Gasdermin D; GSDMD-NT, N-terminal Gasdermin D

    Techniques Used: Activation Assay, Expressing

    Inhibition of P2X7R prevented IS-induced nociceptive behavior and cognitive deficits. a - c Nociceptive behaviors were assessed using periorbital withdrawal threshold and the number of head-scratching within 1 h. ( a ) Baseline periorbital withdrawal thresholds measured before the first drug administration were not significantly different among the four groups. ( b - c ) The sham-VEH, sham-BBG and IS-BBG mice had higher post-treatment periorbital withdrawal thresholds measured 1-h after the last drug administration ( b ) and less head-scratching within 1-h measured immediately after the last drug administration ( c ) than IS-VEH mice, indicating the IS-induced nociceptive behaviors were partially prevented by pretreatment with BBG. d - f Non-spatial recognition memory was assessed using novel object recognition test. ( d ) Schematic illustration of the novel object recognition test. ( e ) All mice showed similar explorations of two identical objects at day 2 (training stage). ( f ) The sham-VEH, sham-BBG and IS-BBG mice showed a higher discrimination ratio than IS-VEH mice, indicating the IS-induced impairment of non-spatial recognition memory was improved by pretreatment with BBG. (Discrimination Ratio = T novel – T old / T novel + T old ). g-i Spatial learning and memory was assessed using Morris water maze test. Average latency to find visible platform on day 1 and hidden platform in target quadrant on day 2–5. All mice exhibited similar average latency to find a visible platform on day 1 ( g ), suggesting the similar vision and motor ability of all mice. From day 2 to day 5 (learning period), the escape latency gradually decreased in all mice and the slope of the decrease was not significantly different among the four groups ( g ), suggesting the similar spatial learning ability of all mice. On day 6 (probe trial), IS-VEH mice displayed a shorter time spent in target quadrant ( h ) and less platform crossings ( i ) than sham-VEH mice, suggesting the impaired spatial memory retention in IS-VEH mice. Although IS-BBG mice displayed a trend with a longer time spent in target quadrant ( h ) and more platform crossings ( i ) than IS-VEH mice, the difference did not reach statistical levels. n = 10 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R prevented IS-induced nociceptive behavior and cognitive deficits. a - c Nociceptive behaviors were assessed using periorbital withdrawal threshold and the number of head-scratching within 1 h. ( a ) Baseline periorbital withdrawal thresholds measured before the first drug administration were not significantly different among the four groups. ( b - c ) The sham-VEH, sham-BBG and IS-BBG mice had higher post-treatment periorbital withdrawal thresholds measured 1-h after the last drug administration ( b ) and less head-scratching within 1-h measured immediately after the last drug administration ( c ) than IS-VEH mice, indicating the IS-induced nociceptive behaviors were partially prevented by pretreatment with BBG. d - f Non-spatial recognition memory was assessed using novel object recognition test. ( d ) Schematic illustration of the novel object recognition test. ( e ) All mice showed similar explorations of two identical objects at day 2 (training stage). ( f ) The sham-VEH, sham-BBG and IS-BBG mice showed a higher discrimination ratio than IS-VEH mice, indicating the IS-induced impairment of non-spatial recognition memory was improved by pretreatment with BBG. (Discrimination Ratio = T novel – T old / T novel + T old ). g-i Spatial learning and memory was assessed using Morris water maze test. Average latency to find visible platform on day 1 and hidden platform in target quadrant on day 2–5. All mice exhibited similar average latency to find a visible platform on day 1 ( g ), suggesting the similar vision and motor ability of all mice. From day 2 to day 5 (learning period), the escape latency gradually decreased in all mice and the slope of the decrease was not significantly different among the four groups ( g ), suggesting the similar spatial learning ability of all mice. On day 6 (probe trial), IS-VEH mice displayed a shorter time spent in target quadrant ( h ) and less platform crossings ( i ) than sham-VEH mice, suggesting the impaired spatial memory retention in IS-VEH mice. Although IS-BBG mice displayed a trend with a longer time spent in target quadrant ( h ) and more platform crossings ( i ) than IS-VEH mice, the difference did not reach statistical levels. n = 10 mice per group. * p

    Techniques Used: Inhibition, Mouse Assay

    Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis. ( a - b ) Representative immunoblots and quantification showed decreased expression levels of P2X7R, NLRP3 (inflammasome receptor) and cleaved caspase-1 (marker of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( c - d ) Representative immunoblots and quantification showed decreased expression levels of GSDMD-NT (pyroptotic marker) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( e – f ) ELISA results showed decreased release of IL-1β and IL-18 (direct downstream products of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 4 mice per group). * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis. ( a - b ) Representative immunoblots and quantification showed decreased expression levels of P2X7R, NLRP3 (inflammasome receptor) and cleaved caspase-1 (marker of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( c - d ) Representative immunoblots and quantification showed decreased expression levels of GSDMD-NT (pyroptotic marker) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( e – f ) ELISA results showed decreased release of IL-1β and IL-18 (direct downstream products of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 4 mice per group). * p

    Techniques Used: Inhibition, Activation Assay, Western Blot, Expressing, Marker, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of P2X7R attenuated IS-induced neuronal loss in the cerebral cortex and hippocampus. ( a - e ) Representative crystal violet images and quantification illustrating increased Nissl-positive neurons in the cerebral cortex of IS-BBG mice compared to IS-VEH mice. The number of Nissl-positive neurons in hippocampal CA1, CA2, CA3 and DG was similar between IS-BBG mice and IS-VEH mice. Magnification × 40. Scale bar, 20 μm. ( f - j ) Representative Luxol fast blue stained images and quantification illustrating no significant difference in white-matter integrity in the corpus callosum (medial), corpus callosum (paramedian), caudoputamen, internal capsule and optic tract among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced neuronal loss in the cerebral cortex and hippocampus. ( a - e ) Representative crystal violet images and quantification illustrating increased Nissl-positive neurons in the cerebral cortex of IS-BBG mice compared to IS-VEH mice. The number of Nissl-positive neurons in hippocampal CA1, CA2, CA3 and DG was similar between IS-BBG mice and IS-VEH mice. Magnification × 40. Scale bar, 20 μm. ( f - j ) Representative Luxol fast blue stained images and quantification illustrating no significant difference in white-matter integrity in the corpus callosum (medial), corpus callosum (paramedian), caudoputamen, internal capsule and optic tract among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Inhibition, Mouse Assay, Staining

    Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis on neurons and microglia in the cerebral cortex and hippocampus. ( a, d ) Double immunofluorescence staining showed a reduction in CC1-positive (marker of inflammasome activation) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. ( b, e ) Double immunofluorescence showed similar CC3-positive (apoptotic marker) neurons (NeuN positive) in the cerebral cortex and hippocampus of the four groups of mice. ( c, f ) Double immunofluorescence staining showed a reduction in GSDMD-positive (pyroptotic marker) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 100. Scale bar, 20 μm. Images were taken under identical exposures and conditions. Abbreviations: IS, inflammatory soup; CC1, cleaved caspase-1; CC3, cleaved caspase-3; GSDMD, Gasdermin D; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; BBG, brilliant blue G; VEH, vehicle
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis on neurons and microglia in the cerebral cortex and hippocampus. ( a, d ) Double immunofluorescence staining showed a reduction in CC1-positive (marker of inflammasome activation) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. ( b, e ) Double immunofluorescence showed similar CC3-positive (apoptotic marker) neurons (NeuN positive) in the cerebral cortex and hippocampus of the four groups of mice. ( c, f ) Double immunofluorescence staining showed a reduction in GSDMD-positive (pyroptotic marker) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 100. Scale bar, 20 μm. Images were taken under identical exposures and conditions. Abbreviations: IS, inflammatory soup; CC1, cleaved caspase-1; CC3, cleaved caspase-3; GSDMD, Gasdermin D; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; BBG, brilliant blue G; VEH, vehicle

    Techniques Used: Inhibition, Activation Assay, Double Immunofluorescence Staining, Marker, Mouse Assay, Immunofluorescence, Binding Assay

    Inhibition of P2X7R attenuated IS-induced gliosis and neuronal loss in the cerebral cortex and hippocampus. ( a, b, e, f ) Representative immunofluorescence and quantification of Iba-1 and GFAP illustrating resistance to microgliosis and astrogliosis due to decreased expression of Iba-1and GFAP in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. ( c, g ) Representative immunofluorescence and quantification of MAP2 illustrating resistance to neuronal loss due to decreased expression of MAP2 in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 20. Scale bar, 20 μm. ( d, h ) Representative immunofluorescence and quantification of MBP illustrating no significant difference in white-matter integrity in the cerebral cortex and hippocampus among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced gliosis and neuronal loss in the cerebral cortex and hippocampus. ( a, b, e, f ) Representative immunofluorescence and quantification of Iba-1 and GFAP illustrating resistance to microgliosis and astrogliosis due to decreased expression of Iba-1and GFAP in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. ( c, g ) Representative immunofluorescence and quantification of MAP2 illustrating resistance to neuronal loss due to decreased expression of MAP2 in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 20. Scale bar, 20 μm. ( d, h ) Representative immunofluorescence and quantification of MBP illustrating no significant difference in white-matter integrity in the cerebral cortex and hippocampus among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Inhibition, Immunofluorescence, Expressing, Mouse Assay

    The experimental protocol of the study. ( a ) Schematic timeline diagram of drug treatment and behavioral assessment in mice. ( b ) Details of the dosing regimen for each group of mice. IS, 20μL/day for 4 days, dural injection; PBS, 20μL/day for 4 days, dural injection; BBG, a specific P2X7R antagonist, 50 mg/kg/day for 4 days, intraperitoneal injection; VEH, 0.9% saline, the same volume as BBG, intraperitoneal injection. Abbreviations: DS, dural stimulation; i.p., intraperitoneal injection; VFT, von Frey’s test; IS, inflammatory soup; PBS, phosphate buffer saline; BBG, brilliant blue G; VEH, vehicle
    Figure Legend Snippet: The experimental protocol of the study. ( a ) Schematic timeline diagram of drug treatment and behavioral assessment in mice. ( b ) Details of the dosing regimen for each group of mice. IS, 20μL/day for 4 days, dural injection; PBS, 20μL/day for 4 days, dural injection; BBG, a specific P2X7R antagonist, 50 mg/kg/day for 4 days, intraperitoneal injection; VEH, 0.9% saline, the same volume as BBG, intraperitoneal injection. Abbreviations: DS, dural stimulation; i.p., intraperitoneal injection; VFT, von Frey’s test; IS, inflammatory soup; PBS, phosphate buffer saline; BBG, brilliant blue G; VEH, vehicle

    Techniques Used: Mouse Assay, Injection

    Repeated dural IS stimulation upregulated P2X7R in the cortical and hippocampal neurons. ( a - b ) Representative immunoblots and quantification illustrated increased levels of P2X7R in the cerebral cortex and hippocampus after repeated dural IS stimulation. ( c - e ) Double immunofluorescence staining showed that P2X7R was co-localized within neurons (NeuN positive) in the cerebral cortex and hippocampus. No substantial colocalization of P2X7R was observed in microglia (Iba-1 positive) and astrocytes (GFAP positive) in the cortex and hippocampus. Magnification × 40. Insets show a higher magnification view (Zoom). Scale bar, 20 μm. Images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Repeated dural IS stimulation upregulated P2X7R in the cortical and hippocampal neurons. ( a - b ) Representative immunoblots and quantification illustrated increased levels of P2X7R in the cerebral cortex and hippocampus after repeated dural IS stimulation. ( c - e ) Double immunofluorescence staining showed that P2X7R was co-localized within neurons (NeuN positive) in the cerebral cortex and hippocampus. No substantial colocalization of P2X7R was observed in microglia (Iba-1 positive) and astrocytes (GFAP positive) in the cortex and hippocampus. Magnification × 40. Insets show a higher magnification view (Zoom). Scale bar, 20 μm. Images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Western Blot, Double Immunofluorescence Staining, Mouse Assay

    12) Product Images from "P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis"

    Article Title: P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis

    Journal: bioRxiv

    doi: 10.1101/2021.12.31.474644

    The effect of P2RX7 inhibition on the primary tumour. C57BL/6J mice were injected with luciferase expressing E0771 cells intra-ductally and were treated intraperitoneal with 10mg/kg A740003 or vehicle controls, daily (n=5 per group). The primary tumour growth was measured using A) bioluminescence–based in vivo imaging and Vernier callipers. B) Representative bioluminescence images of the primary tumours. At the end of the study, the effects of the A740003 treatment was assessed on the excised primary tumours by IHC. The percentage of C) Ki67 positive proliferating cells, D) caspase-3 positive apoptotic cells, E) endomucin positive endothelial cells, and F) F4/80 positive macrophages were measured by QuPath software. G) The percentage of necrotic area in the primary tumours (red outline) was assessed as an average of 3 levels, 100μm apart. The data has been presented as mean ± SD and analysed using unpaired t-test. *P
    Figure Legend Snippet: The effect of P2RX7 inhibition on the primary tumour. C57BL/6J mice were injected with luciferase expressing E0771 cells intra-ductally and were treated intraperitoneal with 10mg/kg A740003 or vehicle controls, daily (n=5 per group). The primary tumour growth was measured using A) bioluminescence–based in vivo imaging and Vernier callipers. B) Representative bioluminescence images of the primary tumours. At the end of the study, the effects of the A740003 treatment was assessed on the excised primary tumours by IHC. The percentage of C) Ki67 positive proliferating cells, D) caspase-3 positive apoptotic cells, E) endomucin positive endothelial cells, and F) F4/80 positive macrophages were measured by QuPath software. G) The percentage of necrotic area in the primary tumours (red outline) was assessed as an average of 3 levels, 100μm apart. The data has been presented as mean ± SD and analysed using unpaired t-test. *P

    Techniques Used: Inhibition, Mouse Assay, Injection, Luciferase, Expressing, In Vivo Imaging, Immunohistochemistry, Software

    The effect of P2RX7 inhibition on the bone microenvironment. A) μCT analyses of osteolytic lesions in tibias of E0771 tumour bearing C57BL/6J mice treated daily with P2RX7 antagonist (10mg/kg A740003) or vehicle controls (n=5 per group); AMC are non-tumour bearing age-matched controls. B) Representative μCT images of proximal tibias from each group. Morphometric analyses of the proximal tibia was done to characterise the structural changes in bone by measuring C) trabecular bone volume fraction (Tr BV/TV), D) trabecular thickness (Tr.Th), E) trabecular number (Tr.N), and F) cortical bone volume. G) Representative 3D μCT images of the tibial trabecular and cortical bone are shown alongside. Historphometric analyses of the tibial trabecular bone was performed by assessing G) osteoblast number per bone perimeter (N.Ob/B.Pm), H) osteoblast surface per bone perimeter (Ob.Pm/B.Pm), I) osteoclast number per bone perimeter (N.Oc/B.Pm), and J) osteoclast surface per bone perimeter (Oc.Pm/B.Pm). K) Representative images of the tibial sections with TRAP-positive osteoclasts (stars) and osteoblasts (arrows) shown on trabecular bone. Scale Bar = 50μm. The data has been presented as mean ± SD and analysed using unpaired t-test. *P
    Figure Legend Snippet: The effect of P2RX7 inhibition on the bone microenvironment. A) μCT analyses of osteolytic lesions in tibias of E0771 tumour bearing C57BL/6J mice treated daily with P2RX7 antagonist (10mg/kg A740003) or vehicle controls (n=5 per group); AMC are non-tumour bearing age-matched controls. B) Representative μCT images of proximal tibias from each group. Morphometric analyses of the proximal tibia was done to characterise the structural changes in bone by measuring C) trabecular bone volume fraction (Tr BV/TV), D) trabecular thickness (Tr.Th), E) trabecular number (Tr.N), and F) cortical bone volume. G) Representative 3D μCT images of the tibial trabecular and cortical bone are shown alongside. Historphometric analyses of the tibial trabecular bone was performed by assessing G) osteoblast number per bone perimeter (N.Ob/B.Pm), H) osteoblast surface per bone perimeter (Ob.Pm/B.Pm), I) osteoclast number per bone perimeter (N.Oc/B.Pm), and J) osteoclast surface per bone perimeter (Oc.Pm/B.Pm). K) Representative images of the tibial sections with TRAP-positive osteoclasts (stars) and osteoblasts (arrows) shown on trabecular bone. Scale Bar = 50μm. The data has been presented as mean ± SD and analysed using unpaired t-test. *P

    Techniques Used: Inhibition, Mouse Assay

    Extracellular vesicles (EVs) from E0771 cells were isolated using size-exclusion columns. A) EVs were enriched in elute fractions 5-8 which were pooled together for subsequent studies. B) size distribution of the isolated EVs as measured by nanoparticle tracking analyses (NTA). C) Western blotting for CD9 and TSG101 as markers for EVs in the cell lysates (CL), EV fractions and post-EV (pEV) fractions. E0771 cells were cultured in hypoxia (1% O 2 ) or normoxia for 8h and EVs isolated subsequently from the conditioned media. D) NTA of the isolated EVs from hypoxic and normoxic cells. E) P2RX7 expression in E0771 cells cultured in normoxic and hypoxic conditions for 8h as assessed by real-time PCR. F) Hif1a and P2RX7 protein expression over time under hypoxic conditions with GAPDH as a loading control. G) The effect of hypoxia on P2RX7 expression was also assessed in primary tumours in vivo . IHC staining for GLUT-1 (as a marker for hypoxia) correlates with P2RX7 in serial sections. H) E0771 cells were treated with varying concentrations of BzATP (0-300μM), and EVs isolated from the media. Western blotting for CD9 and TSG101 shows the effect of P2RX7 activation on EV secretion, with β-Actin serving as a loading control. I) A740003 mediated inhibition of P2RX7 activation by 300μM reduces the EVs (CD9 and TSG101) secreted in the conditioned media. All in vitro data are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using unpaired t-test. ***P
    Figure Legend Snippet: Extracellular vesicles (EVs) from E0771 cells were isolated using size-exclusion columns. A) EVs were enriched in elute fractions 5-8 which were pooled together for subsequent studies. B) size distribution of the isolated EVs as measured by nanoparticle tracking analyses (NTA). C) Western blotting for CD9 and TSG101 as markers for EVs in the cell lysates (CL), EV fractions and post-EV (pEV) fractions. E0771 cells were cultured in hypoxia (1% O 2 ) or normoxia for 8h and EVs isolated subsequently from the conditioned media. D) NTA of the isolated EVs from hypoxic and normoxic cells. E) P2RX7 expression in E0771 cells cultured in normoxic and hypoxic conditions for 8h as assessed by real-time PCR. F) Hif1a and P2RX7 protein expression over time under hypoxic conditions with GAPDH as a loading control. G) The effect of hypoxia on P2RX7 expression was also assessed in primary tumours in vivo . IHC staining for GLUT-1 (as a marker for hypoxia) correlates with P2RX7 in serial sections. H) E0771 cells were treated with varying concentrations of BzATP (0-300μM), and EVs isolated from the media. Western blotting for CD9 and TSG101 shows the effect of P2RX7 activation on EV secretion, with β-Actin serving as a loading control. I) A740003 mediated inhibition of P2RX7 activation by 300μM reduces the EVs (CD9 and TSG101) secreted in the conditioned media. All in vitro data are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using unpaired t-test. ***P

    Techniques Used: Isolation, Western Blot, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, In Vivo, Immunohistochemistry, Staining, Marker, Activation Assay, Inhibition, In Vitro

    A) mRNA expression for P2RX7 in the different molecular subtypes of breast cancer. Data analysed from the TCGA and SCAN-B datasets (n=4669). B) mRNA expression of P2RX7 in triple-negative breast cancer (TNBC, n=317) compared to non-TNBC (n=4119). C) Kaplan-Meier plot for distant-metastasis free survival for patients with low (tercile 1) and high (tercile 3) P2RX7 expression. D) P2RX7 agonist BzATP (100μM) mediated calcium response (green) in E0771 murine breast cancer cells, which is inhibited in presence of a specific antagonist A740003 (10μM, red). Peak intensity and the area-under-curve (AUC) for the calcium response in (D) are presented in (E) and (F) respectively. G) P2RX7 mediated pore formation with higher concentrations of BzATP (300μM) assessed by ethidium bromide uptake in E0771 cells (red), which is inhibited by A740003 (green). P2RX7 over-expressing HEK-293 cells are used as a positive control (blue). (H) Analysis of the AUC for these pore-formation responses. I) Effect of P2RX7 antagonist A740003 on E0771 cell proliferation assessed by WST-1 at 72h. J) The effect of A740003 (10μM) on E0771 migration using scratch assay. All in vitro data is presented relative to the untreated controls and are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using one-way ANOVA. *P
    Figure Legend Snippet: A) mRNA expression for P2RX7 in the different molecular subtypes of breast cancer. Data analysed from the TCGA and SCAN-B datasets (n=4669). B) mRNA expression of P2RX7 in triple-negative breast cancer (TNBC, n=317) compared to non-TNBC (n=4119). C) Kaplan-Meier plot for distant-metastasis free survival for patients with low (tercile 1) and high (tercile 3) P2RX7 expression. D) P2RX7 agonist BzATP (100μM) mediated calcium response (green) in E0771 murine breast cancer cells, which is inhibited in presence of a specific antagonist A740003 (10μM, red). Peak intensity and the area-under-curve (AUC) for the calcium response in (D) are presented in (E) and (F) respectively. G) P2RX7 mediated pore formation with higher concentrations of BzATP (300μM) assessed by ethidium bromide uptake in E0771 cells (red), which is inhibited by A740003 (green). P2RX7 over-expressing HEK-293 cells are used as a positive control (blue). (H) Analysis of the AUC for these pore-formation responses. I) Effect of P2RX7 antagonist A740003 on E0771 cell proliferation assessed by WST-1 at 72h. J) The effect of A740003 (10μM) on E0771 migration using scratch assay. All in vitro data is presented relative to the untreated controls and are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using one-way ANOVA. *P

    Techniques Used: Expressing, Positive Control, Migration, Wound Healing Assay, In Vitro

    13) Product Images from "Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22"

    Article Title: Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2022.910662

    The P2X7R protein is upregulated in nerve terminals of the hippocampus of drug-refractory MTLE-HS human patients. Panel (A) shows that using our methodology nerve terminals isolated from the hippocampus of MTLE-HS patients exhibit a higher density of the synaptic vesicle marker, synaptophysin (~34 kDa), compared to the astrocytic cell marker, GFAP, whilst the opposite was observed in total hippocampal lysates. In panel (B) are shown representative Western blots of the P2X7R immunoreactivity in total lysates and nerve terminals isolated from the human hippocampus of control individuals and MTLE-HS patients; gels were loaded with 100 μg of protein. Two protein species were recognized by the P2X7R antibody from Alomone (#APR-004, Jerusalem, Israel) corresponding to the naturally occurring 67 kDa receptor isoform and to a higher molecular mass (~85 kDa) P2X7R isotype; the latter is highly enriched in nerve terminals of the hippocampus of MTLE-HS patients compared to non-epileptic controls. Please note that the two bands corresponding to the P2X7R protein disappeared after pre-adsorption of the primary antibody with a control antigen peptide equivalent to the amino-acid residues 576–595 of the intracellular C-terminus of the P2X7R (negative control); β-Actin (38–41 kDa) was used as a reference protein. Panel (C) shows computed data obtained from immunoblot experiments; data are expressed as mean ± SD; each individual sample was processed in duplicate; at least three individuals from each group (control and MTLE-HS) were analyzed.
    Figure Legend Snippet: The P2X7R protein is upregulated in nerve terminals of the hippocampus of drug-refractory MTLE-HS human patients. Panel (A) shows that using our methodology nerve terminals isolated from the hippocampus of MTLE-HS patients exhibit a higher density of the synaptic vesicle marker, synaptophysin (~34 kDa), compared to the astrocytic cell marker, GFAP, whilst the opposite was observed in total hippocampal lysates. In panel (B) are shown representative Western blots of the P2X7R immunoreactivity in total lysates and nerve terminals isolated from the human hippocampus of control individuals and MTLE-HS patients; gels were loaded with 100 μg of protein. Two protein species were recognized by the P2X7R antibody from Alomone (#APR-004, Jerusalem, Israel) corresponding to the naturally occurring 67 kDa receptor isoform and to a higher molecular mass (~85 kDa) P2X7R isotype; the latter is highly enriched in nerve terminals of the hippocampus of MTLE-HS patients compared to non-epileptic controls. Please note that the two bands corresponding to the P2X7R protein disappeared after pre-adsorption of the primary antibody with a control antigen peptide equivalent to the amino-acid residues 576–595 of the intracellular C-terminus of the P2X7R (negative control); β-Actin (38–41 kDa) was used as a reference protein. Panel (C) shows computed data obtained from immunoblot experiments; data are expressed as mean ± SD; each individual sample was processed in duplicate; at least three individuals from each group (control and MTLE-HS) were analyzed.

    Techniques Used: Isolation, Marker, Western Blot, Adsorption, Negative Control

    Representative confocal micrographs of different sub-regions of the human hippocampus showing that the P2X7R immunoreactivity (green) is higher in the hippocampus of MTLE-HS patients than of non-epileptic cadaveric controls. A negative control resulting from incubation of the DG/CA4 hippocampal region of an MTLE-HS patient with the anti-rabbit secondary antibody without previous addition of the rabbit anti-P2X7R primary antibody (#APR-004) and differential interference contrast (DIC) images are shown for comparison; three confocal micrographs were obtained per individual; three individuals from each group (control and MTLE-HS) were analyzed showing similar results; scale bars = 300 μm.
    Figure Legend Snippet: Representative confocal micrographs of different sub-regions of the human hippocampus showing that the P2X7R immunoreactivity (green) is higher in the hippocampus of MTLE-HS patients than of non-epileptic cadaveric controls. A negative control resulting from incubation of the DG/CA4 hippocampal region of an MTLE-HS patient with the anti-rabbit secondary antibody without previous addition of the rabbit anti-P2X7R primary antibody (#APR-004) and differential interference contrast (DIC) images are shown for comparison; three confocal micrographs were obtained per individual; three individuals from each group (control and MTLE-HS) were analyzed showing similar results; scale bars = 300 μm.

    Techniques Used: Negative Control, Incubation

    No significant correlation was observed between the P2X7R expression and the age of epileptic patients (panels B and D ) and non-epileptic cadaveric controls (panels A and C ), both in the hippocampus (panels A and B ) and in the temporal neocortex (panels C and D ). Each point represents an individual sample among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. Spearman’s correlation coefficients and significance p values (two-tailed) are shown inside each graph.
    Figure Legend Snippet: No significant correlation was observed between the P2X7R expression and the age of epileptic patients (panels B and D ) and non-epileptic cadaveric controls (panels A and C ), both in the hippocampus (panels A and B ) and in the temporal neocortex (panels C and D ). Each point represents an individual sample among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. Spearman’s correlation coefficients and significance p values (two-tailed) are shown inside each graph.

    Techniques Used: Expressing, Two Tailed Test

    Representative confocal micrographs showing that the P2X7R immunoreactivity is located predominantly in nerve terminals, but not in glial cells, of all sub-regions of the hippocampus of MTLE-HS patients. Synaptic nerve terminals were identified with an antibody against the vesicle-associated membrane protein 1 (VAMP-1 or synaptobrevin 1), whereas astrocytes were stained with an antibody against the glial fibrillary acidic protein (GFAP). Note that VAMP-1-positive nerve terminals (red) are endowed with the P2X7R (green; panel A ), but no significant co-localization was observed between P2X7R (green) and GFAP (red; panel B ); scale bars = 50 μm. Data in panels (C) and (D) correspond to staining overlap and Pearson’s Coefficient (ρ) parameters calculated from three to four confocal micrographs per individual; at least three individuals from each group, control, and MTLE-HS, were analyzed. These parameters were automatically calculated per image with Olympus Fluoview 4.2 Software (Olympus FV1000, Tokyo, Japan) and were used to estimate the co-localization of P2X7R and type-specific cell markers (yellow staining). Overlap between two colors gives values between +1 (total overlap) and 0 (no overlap); the Pearson’s Coefficient (ρ) is a measure of the linear correlation between two variables (stainings), giving values between +1 and −1 inclusive, where 1 is total positive correlation, 0 is no correlation, and −1 is total negative correlation. Higher magnification images show that the P2X7R immunoreactivity also co-localizes with the synaptic vesicle glicoprotein synaptophysin (Synapt), which is one of the most commonly used neuronal cell markers in neuropathology (Panel E ), but not with GFAP (Panel F ). Nuclei are stained with DAPI; cross-reactivity for the secondary antibodies was tested in control experiments in which primary antibodies were omitted (negative controls).
    Figure Legend Snippet: Representative confocal micrographs showing that the P2X7R immunoreactivity is located predominantly in nerve terminals, but not in glial cells, of all sub-regions of the hippocampus of MTLE-HS patients. Synaptic nerve terminals were identified with an antibody against the vesicle-associated membrane protein 1 (VAMP-1 or synaptobrevin 1), whereas astrocytes were stained with an antibody against the glial fibrillary acidic protein (GFAP). Note that VAMP-1-positive nerve terminals (red) are endowed with the P2X7R (green; panel A ), but no significant co-localization was observed between P2X7R (green) and GFAP (red; panel B ); scale bars = 50 μm. Data in panels (C) and (D) correspond to staining overlap and Pearson’s Coefficient (ρ) parameters calculated from three to four confocal micrographs per individual; at least three individuals from each group, control, and MTLE-HS, were analyzed. These parameters were automatically calculated per image with Olympus Fluoview 4.2 Software (Olympus FV1000, Tokyo, Japan) and were used to estimate the co-localization of P2X7R and type-specific cell markers (yellow staining). Overlap between two colors gives values between +1 (total overlap) and 0 (no overlap); the Pearson’s Coefficient (ρ) is a measure of the linear correlation between two variables (stainings), giving values between +1 and −1 inclusive, where 1 is total positive correlation, 0 is no correlation, and −1 is total negative correlation. Higher magnification images show that the P2X7R immunoreactivity also co-localizes with the synaptic vesicle glicoprotein synaptophysin (Synapt), which is one of the most commonly used neuronal cell markers in neuropathology (Panel E ), but not with GFAP (Panel F ). Nuclei are stained with DAPI; cross-reactivity for the secondary antibodies was tested in control experiments in which primary antibodies were omitted (negative controls).

    Techniques Used: Staining, Software

    The inverse relationship between miR-22 serum levels and the P2X7R expression in the hippocampus (A) and temporal neocortex (B) from drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy. Ordinates represent ΔCt variation of P2X7R and miR-22 expression in epileptic patients compared to the corresponding median of non-epileptic controls; 0 represents null variation; positive and negative values correspond to increases and decreases relative to the control population, respectively. Please note that there is a mismatch between miR-22 serum quantifications and P2X7R mRNA determinations in the hippocampus ( A , two patients missing) and temporal neocortex ( B , one patient missing) because RNA samples were insufficient or did not pass the quality assessment.
    Figure Legend Snippet: The inverse relationship between miR-22 serum levels and the P2X7R expression in the hippocampus (A) and temporal neocortex (B) from drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy. Ordinates represent ΔCt variation of P2X7R and miR-22 expression in epileptic patients compared to the corresponding median of non-epileptic controls; 0 represents null variation; positive and negative values correspond to increases and decreases relative to the control population, respectively. Please note that there is a mismatch between miR-22 serum quantifications and P2X7R mRNA determinations in the hippocampus ( A , two patients missing) and temporal neocortex ( B , one patient missing) because RNA samples were insufficient or did not pass the quality assessment.

    Techniques Used: Expressing

    The P2X7R mRNA is overexpressed in the hippocampus and temporal neocortex of drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy compared to non-epileptic cadaveric controls (CTR). qPCR data are expressed as mean ± SD; n numbers inside each bar represent the number of individuals among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. * p
    Figure Legend Snippet: The P2X7R mRNA is overexpressed in the hippocampus and temporal neocortex of drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy compared to non-epileptic cadaveric controls (CTR). qPCR data are expressed as mean ± SD; n numbers inside each bar represent the number of individuals among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. * p

    Techniques Used: Real-time Polymerase Chain Reaction

    14) Product Images from "P2x7 receptors control demyelination and inflammation in the cuprizone model"

    Article Title: P2x7 receptors control demyelination and inflammation in the cuprizone model

    Journal: Brain, Behavior, & Immunity - Health

    doi: 10.1016/j.bbih.2020.100062

    P2x7 receptors are not master regulators of demyelination-associated microglia phenotype. Wild-type and P2x7 knockout mice were treated with cuprizone for 6 weeks and microglial cells purified from the forebrain using flow cytometry. RT-qPCR analysis of inflammasome activationmarkers ( A ) and M1/M2 phenotype genes ( C ) in cell sorted from control and cuprizone-treated mice showed selective up-regulation of several pro-inflammatory genes in demyelination-associated microglia. Data represent relative fold change compared with microglial cells purified from control mice normalized to B2m and Ppia ( n ​= ​3-6 mice per group). ( B , D ) Relative expression of indicate genes in microglia sorted from cuprizone-treated P2x7 knockout mice ( n ​= ​3-6 mice per group; fold change compared with expression levels in cells purified from cuprizone-treated wild-type mice; normalized to B2m and Ppia ). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: P2x7 receptors are not master regulators of demyelination-associated microglia phenotype. Wild-type and P2x7 knockout mice were treated with cuprizone for 6 weeks and microglial cells purified from the forebrain using flow cytometry. RT-qPCR analysis of inflammasome activationmarkers ( A ) and M1/M2 phenotype genes ( C ) in cell sorted from control and cuprizone-treated mice showed selective up-regulation of several pro-inflammatory genes in demyelination-associated microglia. Data represent relative fold change compared with microglial cells purified from control mice normalized to B2m and Ppia ( n ​= ​3-6 mice per group). ( B , D ) Relative expression of indicate genes in microglia sorted from cuprizone-treated P2x7 knockout mice ( n ​= ​3-6 mice per group; fold change compared with expression levels in cells purified from cuprizone-treated wild-type mice; normalized to B2m and Ppia ). CPZ, cuprizone. ∗ p ​

    Techniques Used: Knock-Out, Mouse Assay, Purification, Flow Cytometry, Quantitative RT-PCR, Expressing

    Pharmacological blockade of P2x7 receptors does not improve spontaneous remyelination in the cuprizone model. Mice received cuprizone in the diet for 6 weeks and BBG (50 ​mg/Kg), JNJ-47965567 (JNJ; 30 ​mg/Kg) or vehicle solutions during a 2 week remyelination phase. ( A , B ) LFB and MBP immunostaining revealed extensive demyelination at 6 weeks of cuprizone administration and partial recovery 2 weeks after toxin withdrawal. Scoring of LFB and MBP staining showed no differences between mice treated with BBG ( A ), JNJ-47965567 ( B ) or vehicle during the recovery phase ( n ​= ​4-6 mice per group). ∗∗∗ p ​
    Figure Legend Snippet: Pharmacological blockade of P2x7 receptors does not improve spontaneous remyelination in the cuprizone model. Mice received cuprizone in the diet for 6 weeks and BBG (50 ​mg/Kg), JNJ-47965567 (JNJ; 30 ​mg/Kg) or vehicle solutions during a 2 week remyelination phase. ( A , B ) LFB and MBP immunostaining revealed extensive demyelination at 6 weeks of cuprizone administration and partial recovery 2 weeks after toxin withdrawal. Scoring of LFB and MBP staining showed no differences between mice treated with BBG ( A ), JNJ-47965567 ( B ) or vehicle during the recovery phase ( n ​= ​4-6 mice per group). ∗∗∗ p ​

    Techniques Used: Mouse Assay, Immunostaining, Staining

    Effect of P2x7 receptor antagonists on glial cells and inflammatory responses during remyelination. ( A ) Quantification of CD11b + and Iba1 + cells and GFAP immunoreactivity following treatment with P2x7 receptor antagonists. Treatment with BBG or JNJ-47965567 during recovery from cuprizone intoxication did not modulate the presence of microglia/macrophages or astrocytes in remyelinating corpus callosum ( n ​= ​5-6 mice per group). ( B , C ) Expression of inflammasome- and polarization-related molecules assessed by RT-qPCR in brain tissue from mice treated with BBG ( B ) or JNJ-47965567 ( C ). Data represent fold change relative to vehicle-treated mice normalized to Gapdh , Hprt1 and Ppia ( n ​= ​5-6 mice per group). ∗ p ​
    Figure Legend Snippet: Effect of P2x7 receptor antagonists on glial cells and inflammatory responses during remyelination. ( A ) Quantification of CD11b + and Iba1 + cells and GFAP immunoreactivity following treatment with P2x7 receptor antagonists. Treatment with BBG or JNJ-47965567 during recovery from cuprizone intoxication did not modulate the presence of microglia/macrophages or astrocytes in remyelinating corpus callosum ( n ​= ​5-6 mice per group). ( B , C ) Expression of inflammasome- and polarization-related molecules assessed by RT-qPCR in brain tissue from mice treated with BBG ( B ) or JNJ-47965567 ( C ). Data represent fold change relative to vehicle-treated mice normalized to Gapdh , Hprt1 and Ppia ( n ​= ​5-6 mice per group). ∗ p ​

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR

    Up-regulated P2x7 receptor expression and signalling during cuprizone intoxication. ( A ) RT-qPCR analyses showed significant increases in P2rx7 expression during the time-course of cuprizone administration ( n ​= ​7 mice per group; normalized to Gapdh and Hprt1 . ( B ) Up-regulated P2x7 receptor levels were detected following 6 weeks of cuprizone feeding ( n ​= ​5 mice per group). At each treatment point, brain slices from cuprizone-treated mice were compared with tissue from mice fed a control diet. ( C ) Representative images of P2x7 receptor immunostaining in the corpus callosum of mice treated with a cuprizone containing diet for 6 weeks and untreated controls. Quantification of immunolabelled tissue sections revealed significant up-regulation of P2x7 receptors in demyelinated corpus callosum ( n ​= ​4 mice per group). ( D ) Increased expression of inflammasome-related molecules following 6 weeks of cuprizone intoxication. Transcript levels of indicated genes were assessed by RT-qPCR. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: Up-regulated P2x7 receptor expression and signalling during cuprizone intoxication. ( A ) RT-qPCR analyses showed significant increases in P2rx7 expression during the time-course of cuprizone administration ( n ​= ​7 mice per group; normalized to Gapdh and Hprt1 . ( B ) Up-regulated P2x7 receptor levels were detected following 6 weeks of cuprizone feeding ( n ​= ​5 mice per group). At each treatment point, brain slices from cuprizone-treated mice were compared with tissue from mice fed a control diet. ( C ) Representative images of P2x7 receptor immunostaining in the corpus callosum of mice treated with a cuprizone containing diet for 6 weeks and untreated controls. Quantification of immunolabelled tissue sections revealed significant up-regulation of P2x7 receptors in demyelinated corpus callosum ( n ​= ​4 mice per group). ( D ) Increased expression of inflammasome-related molecules following 6 weeks of cuprizone intoxication. Transcript levels of indicated genes were assessed by RT-qPCR. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). CPZ, cuprizone. ∗ p ​

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Immunostaining

    P2x7 receptor deficient mice are resistant to cuprizone-induced demyelination. ( A ) P2x7 +/+ and P2x7 -/- mice were fed 0.3% cuprizone in the diet for 6 weeks. Analysis of tissue sections stained for LFB and MBP showed attenuated myelin loss in P2x7 receptor deficient mice ( n ​= ​3-5 mice). Representative images depict the loss of MBP immunolabelling after cuprizone administration in P2x7 +/+ and P2x7 -/- mice. ( B-D ) The corpus callosum was immunostained for CD11b, GFAP, NG2, CD3, MAC387 and myeloperoxidase (MPO) as markers of microglia/macrophages, astrocytes, OPCs, T cells, recently infiltrating monocytes/macrophages and neutrophils, respectively. Quantification and representative images show a reduced accumulation of astrocytes and microglia in P2x7 -/- mice at 6 weeks of cuprizone diet ( B ), but no differences regarding the recruitment of OPCs ( C ) or peripheral immune cells ( D ) to demyelinated tissue ( n ​= ​4-5 mice). ( E ) Transcript levels of inflammasome-associated genes were assessed by RT-qPCR in brain tissue from P2x7 +/+ and P2x7 -/- mice at 6 weeks of cuprizone intoxication. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). Scale bars: 200 ​μm ( A ) and 50 ​μm ( B ). CPZ, cuprizone; N.D., not detected. ∗ p ​
    Figure Legend Snippet: P2x7 receptor deficient mice are resistant to cuprizone-induced demyelination. ( A ) P2x7 +/+ and P2x7 -/- mice were fed 0.3% cuprizone in the diet for 6 weeks. Analysis of tissue sections stained for LFB and MBP showed attenuated myelin loss in P2x7 receptor deficient mice ( n ​= ​3-5 mice). Representative images depict the loss of MBP immunolabelling after cuprizone administration in P2x7 +/+ and P2x7 -/- mice. ( B-D ) The corpus callosum was immunostained for CD11b, GFAP, NG2, CD3, MAC387 and myeloperoxidase (MPO) as markers of microglia/macrophages, astrocytes, OPCs, T cells, recently infiltrating monocytes/macrophages and neutrophils, respectively. Quantification and representative images show a reduced accumulation of astrocytes and microglia in P2x7 -/- mice at 6 weeks of cuprizone diet ( B ), but no differences regarding the recruitment of OPCs ( C ) or peripheral immune cells ( D ) to demyelinated tissue ( n ​= ​4-5 mice). ( E ) Transcript levels of inflammasome-associated genes were assessed by RT-qPCR in brain tissue from P2x7 +/+ and P2x7 -/- mice at 6 weeks of cuprizone intoxication. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). Scale bars: 200 ​μm ( A ) and 50 ​μm ( B ). CPZ, cuprizone; N.D., not detected. ∗ p ​

    Techniques Used: Mouse Assay, Staining, Quantitative RT-PCR

    P2x7 receptors promote the accumulation of pro-inflammatory microglia during cuprizone-induced demyelination. ( A ) RT-qPCR analysis of M1 and M2 signature genes in brain tissue from mice treated with a cuprizone-containing diet for 6 weeks. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). ( B ) P2x7 knockout mice displayed down-regulated induction of M1 phenotype markers at 6 weeks of cuprizone intoxication. Results are expressed as relative fold change relative to controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​6 mice per group). ( C-D ) Quantification and representative images depicting iNOS and Arg-1 colocalization with CD11b in mice treated with cuprizone for 6 weeks. P2x7 -/- mice exhibited a significant reduction in the number of CD11b + cells expressing iNOS in demyelinated corpus callosum. Scale bars: 20 ​μm. CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: P2x7 receptors promote the accumulation of pro-inflammatory microglia during cuprizone-induced demyelination. ( A ) RT-qPCR analysis of M1 and M2 signature genes in brain tissue from mice treated with a cuprizone-containing diet for 6 weeks. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​4-5 mice per group). ( B ) P2x7 knockout mice displayed down-regulated induction of M1 phenotype markers at 6 weeks of cuprizone intoxication. Results are expressed as relative fold change relative to controls normalized to Gapdh, Hprt1 and Ppia ( n ​= ​6 mice per group). ( C-D ) Quantification and representative images depicting iNOS and Arg-1 colocalization with CD11b in mice treated with cuprizone for 6 weeks. P2x7 -/- mice exhibited a significant reduction in the number of CD11b + cells expressing iNOS in demyelinated corpus callosum. Scale bars: 20 ​μm. CPZ, cuprizone. ∗ p ​

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Knock-Out, Expressing

    Cell-specific analysis of P2x7 receptor expression in cuprizone-treated mice. Confocal images corresponding to double immunostaining of P2x7 receptors and the microglia/macrophage marker CD11b ( A ) and the astrocytic marker GFAP ( B ) in the corpus callosum of mice fed a cuprizone containing diet for 6 weeks. ( C-D ) Analysis of confocal fluorescent images indicated higher colocalization of P2x7 receptor immunopositive puncta with CD11b + microglia than with GFAP + astrocytic profiles, both in control and in cuprizone-treated mice ( n ​= ​4 mice per group). CPZ, cuprizone. ∗ p ​
    Figure Legend Snippet: Cell-specific analysis of P2x7 receptor expression in cuprizone-treated mice. Confocal images corresponding to double immunostaining of P2x7 receptors and the microglia/macrophage marker CD11b ( A ) and the astrocytic marker GFAP ( B ) in the corpus callosum of mice fed a cuprizone containing diet for 6 weeks. ( C-D ) Analysis of confocal fluorescent images indicated higher colocalization of P2x7 receptor immunopositive puncta with CD11b + microglia than with GFAP + astrocytic profiles, both in control and in cuprizone-treated mice ( n ​= ​4 mice per group). CPZ, cuprizone. ∗ p ​

    Techniques Used: Expressing, Mouse Assay, Double Immunostaining, Marker

    15) Product Images from "P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine"

    Article Title: P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine

    Journal: The Journal of Headache and Pain

    doi: 10.1186/s10194-022-01442-8

    Schematic diagram for the mechanisms of P2X7R in the regulation of inflammasome priming, activation and programmed cell death pathways in the brain following repeated dural IS stimulation. The expression of P2X7R is upregulated after repeated dural IS stimulation. P2X7R can activate the assembly of NLRP3 inflammasome, a multi-protein complex including NLRP3, adaptor (i.e., ASC) and effector proteins (i.e., total caspase-1). The formation of an inflammasome complex activates and converts total caspase-1 into active cleaved caspase-1. There are three main groups of substrates that are targeted by cleaved caspase-1. Firstly, cleaved caspase-1 can cleave both precursor IL-1β and IL-18 into active proinflammatory cytokines, mature IL-1β and IL-18. Secondly, cleaved ca spase-1 can cleave GSDMD-FL into GSDMD-NT that self-oligomerize onto the plasma membrane to form a pore to facilitate the influx of water molecules to induce a lytic form of cell death known as pyroptosis. Thirdly, cleaved caspase-1 can cleave and activate total caspase-3 into cleave caspase-3 to induce apoptosis. Abbreviations: GSDMD-FL, full length Gasdermin D; GSDMD-NT, N-terminal Gasdermin D
    Figure Legend Snippet: Schematic diagram for the mechanisms of P2X7R in the regulation of inflammasome priming, activation and programmed cell death pathways in the brain following repeated dural IS stimulation. The expression of P2X7R is upregulated after repeated dural IS stimulation. P2X7R can activate the assembly of NLRP3 inflammasome, a multi-protein complex including NLRP3, adaptor (i.e., ASC) and effector proteins (i.e., total caspase-1). The formation of an inflammasome complex activates and converts total caspase-1 into active cleaved caspase-1. There are three main groups of substrates that are targeted by cleaved caspase-1. Firstly, cleaved caspase-1 can cleave both precursor IL-1β and IL-18 into active proinflammatory cytokines, mature IL-1β and IL-18. Secondly, cleaved ca spase-1 can cleave GSDMD-FL into GSDMD-NT that self-oligomerize onto the plasma membrane to form a pore to facilitate the influx of water molecules to induce a lytic form of cell death known as pyroptosis. Thirdly, cleaved caspase-1 can cleave and activate total caspase-3 into cleave caspase-3 to induce apoptosis. Abbreviations: GSDMD-FL, full length Gasdermin D; GSDMD-NT, N-terminal Gasdermin D

    Techniques Used: Activation Assay, Expressing

    Inhibition of P2X7R prevented IS-induced nociceptive behavior and cognitive deficits. a - c Nociceptive behaviors were assessed using periorbital withdrawal threshold and the number of head-scratching within 1 h. ( a ) Baseline periorbital withdrawal thresholds measured before the first drug administration were not significantly different among the four groups. ( b - c ) The sham-VEH, sham-BBG and IS-BBG mice had higher post-treatment periorbital withdrawal thresholds measured 1-h after the last drug administration ( b ) and less head-scratching within 1-h measured immediately after the last drug administration ( c ) than IS-VEH mice, indicating the IS-induced nociceptive behaviors were partially prevented by pretreatment with BBG. d - f Non-spatial recognition memory was assessed using novel object recognition test. ( d ) Schematic illustration of the novel object recognition test. ( e ) All mice showed similar explorations of two identical objects at day 2 (training stage). ( f ) The sham-VEH, sham-BBG and IS-BBG mice showed a higher discrimination ratio than IS-VEH mice, indicating the IS-induced impairment of non-spatial recognition memory was improved by pretreatment with BBG. (Discrimination Ratio = T novel – T old / T novel + T old ). g-i Spatial learning and memory was assessed using Morris water maze test. Average latency to find visible platform on day 1 and hidden platform in target quadrant on day 2–5. All mice exhibited similar average latency to find a visible platform on day 1 ( g ), suggesting the similar vision and motor ability of all mice. From day 2 to day 5 (learning period), the escape latency gradually decreased in all mice and the slope of the decrease was not significantly different among the four groups ( g ), suggesting the similar spatial learning ability of all mice. On day 6 (probe trial), IS-VEH mice displayed a shorter time spent in target quadrant ( h ) and less platform crossings ( i ) than sham-VEH mice, suggesting the impaired spatial memory retention in IS-VEH mice. Although IS-BBG mice displayed a trend with a longer time spent in target quadrant ( h ) and more platform crossings ( i ) than IS-VEH mice, the difference did not reach statistical levels. n = 10 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R prevented IS-induced nociceptive behavior and cognitive deficits. a - c Nociceptive behaviors were assessed using periorbital withdrawal threshold and the number of head-scratching within 1 h. ( a ) Baseline periorbital withdrawal thresholds measured before the first drug administration were not significantly different among the four groups. ( b - c ) The sham-VEH, sham-BBG and IS-BBG mice had higher post-treatment periorbital withdrawal thresholds measured 1-h after the last drug administration ( b ) and less head-scratching within 1-h measured immediately after the last drug administration ( c ) than IS-VEH mice, indicating the IS-induced nociceptive behaviors were partially prevented by pretreatment with BBG. d - f Non-spatial recognition memory was assessed using novel object recognition test. ( d ) Schematic illustration of the novel object recognition test. ( e ) All mice showed similar explorations of two identical objects at day 2 (training stage). ( f ) The sham-VEH, sham-BBG and IS-BBG mice showed a higher discrimination ratio than IS-VEH mice, indicating the IS-induced impairment of non-spatial recognition memory was improved by pretreatment with BBG. (Discrimination Ratio = T novel – T old / T novel + T old ). g-i Spatial learning and memory was assessed using Morris water maze test. Average latency to find visible platform on day 1 and hidden platform in target quadrant on day 2–5. All mice exhibited similar average latency to find a visible platform on day 1 ( g ), suggesting the similar vision and motor ability of all mice. From day 2 to day 5 (learning period), the escape latency gradually decreased in all mice and the slope of the decrease was not significantly different among the four groups ( g ), suggesting the similar spatial learning ability of all mice. On day 6 (probe trial), IS-VEH mice displayed a shorter time spent in target quadrant ( h ) and less platform crossings ( i ) than sham-VEH mice, suggesting the impaired spatial memory retention in IS-VEH mice. Although IS-BBG mice displayed a trend with a longer time spent in target quadrant ( h ) and more platform crossings ( i ) than IS-VEH mice, the difference did not reach statistical levels. n = 10 mice per group. * p

    Techniques Used: Inhibition, Mouse Assay

    Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis. ( a - b ) Representative immunoblots and quantification showed decreased expression levels of P2X7R, NLRP3 (inflammasome receptor) and cleaved caspase-1 (marker of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( c - d ) Representative immunoblots and quantification showed decreased expression levels of GSDMD-NT (pyroptotic marker) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( e – f ) ELISA results showed decreased release of IL-1β and IL-18 (direct downstream products of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 4 mice per group). * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis. ( a - b ) Representative immunoblots and quantification showed decreased expression levels of P2X7R, NLRP3 (inflammasome receptor) and cleaved caspase-1 (marker of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( c - d ) Representative immunoblots and quantification showed decreased expression levels of GSDMD-NT (pyroptotic marker) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 6 mice per group). ( e – f ) ELISA results showed decreased release of IL-1β and IL-18 (direct downstream products of inflammasome activation) in the cerebral cortex of IS-BBG mice compared to IS-VEH mice ( n = 4 mice per group). * p

    Techniques Used: Inhibition, Activation Assay, Western Blot, Expressing, Marker, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of P2X7R attenuated IS-induced neuronal loss in the cerebral cortex and hippocampus. ( a - e ) Representative crystal violet images and quantification illustrating increased Nissl-positive neurons in the cerebral cortex of IS-BBG mice compared to IS-VEH mice. The number of Nissl-positive neurons in hippocampal CA1, CA2, CA3 and DG was similar between IS-BBG mice and IS-VEH mice. Magnification × 40. Scale bar, 20 μm. ( f - j ) Representative Luxol fast blue stained images and quantification illustrating no significant difference in white-matter integrity in the corpus callosum (medial), corpus callosum (paramedian), caudoputamen, internal capsule and optic tract among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced neuronal loss in the cerebral cortex and hippocampus. ( a - e ) Representative crystal violet images and quantification illustrating increased Nissl-positive neurons in the cerebral cortex of IS-BBG mice compared to IS-VEH mice. The number of Nissl-positive neurons in hippocampal CA1, CA2, CA3 and DG was similar between IS-BBG mice and IS-VEH mice. Magnification × 40. Scale bar, 20 μm. ( f - j ) Representative Luxol fast blue stained images and quantification illustrating no significant difference in white-matter integrity in the corpus callosum (medial), corpus callosum (paramedian), caudoputamen, internal capsule and optic tract among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Inhibition, Mouse Assay, Staining

    Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis on neurons and microglia in the cerebral cortex and hippocampus. ( a, d ) Double immunofluorescence staining showed a reduction in CC1-positive (marker of inflammasome activation) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. ( b, e ) Double immunofluorescence showed similar CC3-positive (apoptotic marker) neurons (NeuN positive) in the cerebral cortex and hippocampus of the four groups of mice. ( c, f ) Double immunofluorescence staining showed a reduction in GSDMD-positive (pyroptotic marker) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 100. Scale bar, 20 μm. Images were taken under identical exposures and conditions. Abbreviations: IS, inflammatory soup; CC1, cleaved caspase-1; CC3, cleaved caspase-3; GSDMD, Gasdermin D; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; BBG, brilliant blue G; VEH, vehicle
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced NLRP3 inflammasome activation and pyroptosis on neurons and microglia in the cerebral cortex and hippocampus. ( a, d ) Double immunofluorescence staining showed a reduction in CC1-positive (marker of inflammasome activation) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. ( b, e ) Double immunofluorescence showed similar CC3-positive (apoptotic marker) neurons (NeuN positive) in the cerebral cortex and hippocampus of the four groups of mice. ( c, f ) Double immunofluorescence staining showed a reduction in GSDMD-positive (pyroptotic marker) neurons (NeuN positive) and microglia (Iba-1 positive) in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 100. Scale bar, 20 μm. Images were taken under identical exposures and conditions. Abbreviations: IS, inflammatory soup; CC1, cleaved caspase-1; CC3, cleaved caspase-3; GSDMD, Gasdermin D; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; BBG, brilliant blue G; VEH, vehicle

    Techniques Used: Inhibition, Activation Assay, Double Immunofluorescence Staining, Marker, Mouse Assay, Immunofluorescence, Binding Assay

    Inhibition of P2X7R attenuated IS-induced gliosis and neuronal loss in the cerebral cortex and hippocampus. ( a, b, e, f ) Representative immunofluorescence and quantification of Iba-1 and GFAP illustrating resistance to microgliosis and astrogliosis due to decreased expression of Iba-1and GFAP in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. ( c, g ) Representative immunofluorescence and quantification of MAP2 illustrating resistance to neuronal loss due to decreased expression of MAP2 in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 20. Scale bar, 20 μm. ( d, h ) Representative immunofluorescence and quantification of MBP illustrating no significant difference in white-matter integrity in the cerebral cortex and hippocampus among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Inhibition of P2X7R attenuated IS-induced gliosis and neuronal loss in the cerebral cortex and hippocampus. ( a, b, e, f ) Representative immunofluorescence and quantification of Iba-1 and GFAP illustrating resistance to microgliosis and astrogliosis due to decreased expression of Iba-1and GFAP in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. ( c, g ) Representative immunofluorescence and quantification of MAP2 illustrating resistance to neuronal loss due to decreased expression of MAP2 in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 20. Scale bar, 20 μm. ( d, h ) Representative immunofluorescence and quantification of MBP illustrating no significant difference in white-matter integrity in the cerebral cortex and hippocampus among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Inhibition, Immunofluorescence, Expressing, Mouse Assay

    The experimental protocol of the study. ( a ) Schematic timeline diagram of drug treatment and behavioral assessment in mice. ( b ) Details of the dosing regimen for each group of mice. IS, 20μL/day for 4 days, dural injection; PBS, 20μL/day for 4 days, dural injection; BBG, a specific P2X7R antagonist, 50 mg/kg/day for 4 days, intraperitoneal injection; VEH, 0.9% saline, the same volume as BBG, intraperitoneal injection. Abbreviations: DS, dural stimulation; i.p., intraperitoneal injection; VFT, von Frey’s test; IS, inflammatory soup; PBS, phosphate buffer saline; BBG, brilliant blue G; VEH, vehicle
    Figure Legend Snippet: The experimental protocol of the study. ( a ) Schematic timeline diagram of drug treatment and behavioral assessment in mice. ( b ) Details of the dosing regimen for each group of mice. IS, 20μL/day for 4 days, dural injection; PBS, 20μL/day for 4 days, dural injection; BBG, a specific P2X7R antagonist, 50 mg/kg/day for 4 days, intraperitoneal injection; VEH, 0.9% saline, the same volume as BBG, intraperitoneal injection. Abbreviations: DS, dural stimulation; i.p., intraperitoneal injection; VFT, von Frey’s test; IS, inflammatory soup; PBS, phosphate buffer saline; BBG, brilliant blue G; VEH, vehicle

    Techniques Used: Mouse Assay, Injection

    Repeated dural IS stimulation upregulated P2X7R in the cortical and hippocampal neurons. ( a - b ) Representative immunoblots and quantification illustrated increased levels of P2X7R in the cerebral cortex and hippocampus after repeated dural IS stimulation. ( c - e ) Double immunofluorescence staining showed that P2X7R was co-localized within neurons (NeuN positive) in the cerebral cortex and hippocampus. No substantial colocalization of P2X7R was observed in microglia (Iba-1 positive) and astrocytes (GFAP positive) in the cortex and hippocampus. Magnification × 40. Insets show a higher magnification view (Zoom). Scale bar, 20 μm. Images were taken under identical exposures and conditions. n = 6 mice per group. * p
    Figure Legend Snippet: Repeated dural IS stimulation upregulated P2X7R in the cortical and hippocampal neurons. ( a - b ) Representative immunoblots and quantification illustrated increased levels of P2X7R in the cerebral cortex and hippocampus after repeated dural IS stimulation. ( c - e ) Double immunofluorescence staining showed that P2X7R was co-localized within neurons (NeuN positive) in the cerebral cortex and hippocampus. No substantial colocalization of P2X7R was observed in microglia (Iba-1 positive) and astrocytes (GFAP positive) in the cortex and hippocampus. Magnification × 40. Insets show a higher magnification view (Zoom). Scale bar, 20 μm. Images were taken under identical exposures and conditions. n = 6 mice per group. * p

    Techniques Used: Western Blot, Double Immunofluorescence Staining, Mouse Assay

    16) Product Images from "Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells"

    Article Title: Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2021.793769

    Detection of the P2X7R in hiPSC-derived neuronal and microglia-like cell cultures. Immunocytochemical detection of P2X7R in different differentiation stages of control and fAD hiPSC-derived neurons (A) show the presence of the P2X7 receptor (green) in early stages of neuronal differentiation (NPC and TD07) and weaker signal in later differentiation stages (TD35, TD63). In contrast, a strong signal was detected on hiPSC-derived microglia (B) in both the early stage of microglial maturation in monoculture, as well as in co-culture with neurons (both control and fAD). Detection of coexpression of P2X7R (green) with GFAP (red) positive astrocytes (C) . The pictures are representative of at least three experiments performed on three independent cell cultures. For Western blot analysis 10 μg of protein was loaded on the gel. Representative pictures of the results (D) show the presence of a positive signal for P2X7R protein in all neuronal samples, with signal intensity decreasing along with the differentiation time points (D) . Densitometric analysis of the Western blot detection illustrates the decrease in signal intensity. The analysis did not show a significant difference between the control and fAD cell line (E) . Western blot analysis was performed in biological triplicates and analyzed in at least three technical replicates. Error bars represent the mean ± SD of three measurements.
    Figure Legend Snippet: Detection of the P2X7R in hiPSC-derived neuronal and microglia-like cell cultures. Immunocytochemical detection of P2X7R in different differentiation stages of control and fAD hiPSC-derived neurons (A) show the presence of the P2X7 receptor (green) in early stages of neuronal differentiation (NPC and TD07) and weaker signal in later differentiation stages (TD35, TD63). In contrast, a strong signal was detected on hiPSC-derived microglia (B) in both the early stage of microglial maturation in monoculture, as well as in co-culture with neurons (both control and fAD). Detection of coexpression of P2X7R (green) with GFAP (red) positive astrocytes (C) . The pictures are representative of at least three experiments performed on three independent cell cultures. For Western blot analysis 10 μg of protein was loaded on the gel. Representative pictures of the results (D) show the presence of a positive signal for P2X7R protein in all neuronal samples, with signal intensity decreasing along with the differentiation time points (D) . Densitometric analysis of the Western blot detection illustrates the decrease in signal intensity. The analysis did not show a significant difference between the control and fAD cell line (E) . Western blot analysis was performed in biological triplicates and analyzed in at least three technical replicates. Error bars represent the mean ± SD of three measurements.

    Techniques Used: Derivative Assay, Co-Culture Assay, Western Blot

    Representative confocal images suggest intracellular localization of the P2X7 receptor’s signal (in green) in Ctrl and fAD neuronal cells (NPC or neurons; labeled with TUBB3 (in red) (A) and membranous localization in microglia-like cells (B) . Each staining is a representative picture of at least six independent experiments. Microglia-like cells were co-stained with IBA1 (red). Scale bar: 20 μm (C,D) and (E) show representative Western blot results of biotinylation analysis. Arrows indicate the bands representing the canonical ≈72 kDa sized P2X7R localized in the IC (intra-cellular) fraction in the case of Ctrl and fAD neurons’ (TD53) samples (D) and the M (membrane) fraction of microglia samples (E) and both fractions in NPC samples (C) . Red arrows indicate the presence of the P2X7R band in the M fractions. The Integrin α7 (C,D) and CD11c (E) are plasma membrane proteins present only in the M factions. HSP27 is a nuclear protein present only in the IC fractions (C–E) . Integrin α7 and HSP27 were used as controls of the efficiency of biotinylation and the separation of biotinylated proteins. Western blot measurements were per-formed as biological triplicates and a duplicate in the case of microglia-like cells. Ctrl, control; Neu, neuron; NPC, neuronal progenitor cell; IC, intra-cellular; M, membrane.
    Figure Legend Snippet: Representative confocal images suggest intracellular localization of the P2X7 receptor’s signal (in green) in Ctrl and fAD neuronal cells (NPC or neurons; labeled with TUBB3 (in red) (A) and membranous localization in microglia-like cells (B) . Each staining is a representative picture of at least six independent experiments. Microglia-like cells were co-stained with IBA1 (red). Scale bar: 20 μm (C,D) and (E) show representative Western blot results of biotinylation analysis. Arrows indicate the bands representing the canonical ≈72 kDa sized P2X7R localized in the IC (intra-cellular) fraction in the case of Ctrl and fAD neurons’ (TD53) samples (D) and the M (membrane) fraction of microglia samples (E) and both fractions in NPC samples (C) . Red arrows indicate the presence of the P2X7R band in the M fractions. The Integrin α7 (C,D) and CD11c (E) are plasma membrane proteins present only in the M factions. HSP27 is a nuclear protein present only in the IC fractions (C–E) . Integrin α7 and HSP27 were used as controls of the efficiency of biotinylation and the separation of biotinylated proteins. Western blot measurements were per-formed as biological triplicates and a duplicate in the case of microglia-like cells. Ctrl, control; Neu, neuron; NPC, neuronal progenitor cell; IC, intra-cellular; M, membrane.

    Techniques Used: Labeling, Staining, Western Blot

    Functional assay results after the application of P2X7R agonists ATP and BzATP alone upon pre-incubation with the highly specific P2X7R antagonist JNJ 47965567. The working concentration of JNJ 47965567 was 1 μM in all conditions. (A) Outline of the experimental design. Cells were treated for 24 h with ATP or BzATP in different concentrations to promote P2X7R activation; in the samples treated with the P2X7R antagonist JNJ 47965567, the pre-treatment was performed for 30 min before the application of ATP or BzATP to induce blockage of the receptor. Cell cultures were monitored as representative phase-contrast photographs show. Cell viability was measured using PrestoBlue cell viability assay after the treatment in control (B,C) and fAD neuronal pro-genitors (D,E) ; TD35 differentiation stage control (F,G) and fAD neuronal cells (H,I) and microglia-like cells differentiated for 2 weeks (J,K) . In all graphs, the viability of the untreated cells represents 100% viability, and the viability of positive controls (cells treated with water to induce total cell death) represent 0% viability. All the measured values were normalized to the two controls. The presented data are of two independent experiments with each condition performed in triplicate cultures. Error bars represent the mean ± SD. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test; p -value ***
    Figure Legend Snippet: Functional assay results after the application of P2X7R agonists ATP and BzATP alone upon pre-incubation with the highly specific P2X7R antagonist JNJ 47965567. The working concentration of JNJ 47965567 was 1 μM in all conditions. (A) Outline of the experimental design. Cells were treated for 24 h with ATP or BzATP in different concentrations to promote P2X7R activation; in the samples treated with the P2X7R antagonist JNJ 47965567, the pre-treatment was performed for 30 min before the application of ATP or BzATP to induce blockage of the receptor. Cell cultures were monitored as representative phase-contrast photographs show. Cell viability was measured using PrestoBlue cell viability assay after the treatment in control (B,C) and fAD neuronal pro-genitors (D,E) ; TD35 differentiation stage control (F,G) and fAD neuronal cells (H,I) and microglia-like cells differentiated for 2 weeks (J,K) . In all graphs, the viability of the untreated cells represents 100% viability, and the viability of positive controls (cells treated with water to induce total cell death) represent 0% viability. All the measured values were normalized to the two controls. The presented data are of two independent experiments with each condition performed in triplicate cultures. Error bars represent the mean ± SD. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test; p -value ***

    Techniques Used: Functional Assay, Incubation, Concentration Assay, Activation Assay, Viability Assay

    17) Product Images from "Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22"

    Article Title: Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2022.910662

    The P2X7R protein is upregulated in nerve terminals of the hippocampus of drug-refractory MTLE-HS human patients. Panel (A) shows that using our methodology nerve terminals isolated from the hippocampus of MTLE-HS patients exhibit a higher density of the synaptic vesicle marker, synaptophysin (~34 kDa), compared to the astrocytic cell marker, GFAP, whilst the opposite was observed in total hippocampal lysates. In panel (B) are shown representative Western blots of the P2X7R immunoreactivity in total lysates and nerve terminals isolated from the human hippocampus of control individuals and MTLE-HS patients; gels were loaded with 100 μg of protein. Two protein species were recognized by the P2X7R antibody from Alomone (#APR-004, Jerusalem, Israel) corresponding to the naturally occurring 67 kDa receptor isoform and to a higher molecular mass (~85 kDa) P2X7R isotype; the latter is highly enriched in nerve terminals of the hippocampus of MTLE-HS patients compared to non-epileptic controls. Please note that the two bands corresponding to the P2X7R protein disappeared after pre-adsorption of the primary antibody with a control antigen peptide equivalent to the amino-acid residues 576–595 of the intracellular C-terminus of the P2X7R (negative control); β-Actin (38–41 kDa) was used as a reference protein. Panel (C) shows computed data obtained from immunoblot experiments; data are expressed as mean ± SD; each individual sample was processed in duplicate; at least three individuals from each group (control and MTLE-HS) were analyzed.
    Figure Legend Snippet: The P2X7R protein is upregulated in nerve terminals of the hippocampus of drug-refractory MTLE-HS human patients. Panel (A) shows that using our methodology nerve terminals isolated from the hippocampus of MTLE-HS patients exhibit a higher density of the synaptic vesicle marker, synaptophysin (~34 kDa), compared to the astrocytic cell marker, GFAP, whilst the opposite was observed in total hippocampal lysates. In panel (B) are shown representative Western blots of the P2X7R immunoreactivity in total lysates and nerve terminals isolated from the human hippocampus of control individuals and MTLE-HS patients; gels were loaded with 100 μg of protein. Two protein species were recognized by the P2X7R antibody from Alomone (#APR-004, Jerusalem, Israel) corresponding to the naturally occurring 67 kDa receptor isoform and to a higher molecular mass (~85 kDa) P2X7R isotype; the latter is highly enriched in nerve terminals of the hippocampus of MTLE-HS patients compared to non-epileptic controls. Please note that the two bands corresponding to the P2X7R protein disappeared after pre-adsorption of the primary antibody with a control antigen peptide equivalent to the amino-acid residues 576–595 of the intracellular C-terminus of the P2X7R (negative control); β-Actin (38–41 kDa) was used as a reference protein. Panel (C) shows computed data obtained from immunoblot experiments; data are expressed as mean ± SD; each individual sample was processed in duplicate; at least three individuals from each group (control and MTLE-HS) were analyzed.

    Techniques Used: Isolation, Marker, Western Blot, Adsorption, Negative Control

    Representative confocal micrographs of different sub-regions of the human hippocampus showing that the P2X7R immunoreactivity (green) is higher in the hippocampus of MTLE-HS patients than of non-epileptic cadaveric controls. A negative control resulting from incubation of the DG/CA4 hippocampal region of an MTLE-HS patient with the anti-rabbit secondary antibody without previous addition of the rabbit anti-P2X7R primary antibody (#APR-004) and differential interference contrast (DIC) images are shown for comparison; three confocal micrographs were obtained per individual; three individuals from each group (control and MTLE-HS) were analyzed showing similar results; scale bars = 300 μm.
    Figure Legend Snippet: Representative confocal micrographs of different sub-regions of the human hippocampus showing that the P2X7R immunoreactivity (green) is higher in the hippocampus of MTLE-HS patients than of non-epileptic cadaveric controls. A negative control resulting from incubation of the DG/CA4 hippocampal region of an MTLE-HS patient with the anti-rabbit secondary antibody without previous addition of the rabbit anti-P2X7R primary antibody (#APR-004) and differential interference contrast (DIC) images are shown for comparison; three confocal micrographs were obtained per individual; three individuals from each group (control and MTLE-HS) were analyzed showing similar results; scale bars = 300 μm.

    Techniques Used: Negative Control, Incubation

    No significant correlation was observed between the P2X7R expression and the age of epileptic patients (panels B and D ) and non-epileptic cadaveric controls (panels A and C ), both in the hippocampus (panels A and B ) and in the temporal neocortex (panels C and D ). Each point represents an individual sample among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. Spearman’s correlation coefficients and significance p values (two-tailed) are shown inside each graph.
    Figure Legend Snippet: No significant correlation was observed between the P2X7R expression and the age of epileptic patients (panels B and D ) and non-epileptic cadaveric controls (panels A and C ), both in the hippocampus (panels A and B ) and in the temporal neocortex (panels C and D ). Each point represents an individual sample among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. Spearman’s correlation coefficients and significance p values (two-tailed) are shown inside each graph.

    Techniques Used: Expressing, Two Tailed Test

    Representative confocal micrographs showing that the P2X7R immunoreactivity is located predominantly in nerve terminals, but not in glial cells, of all sub-regions of the hippocampus of MTLE-HS patients. Synaptic nerve terminals were identified with an antibody against the vesicle-associated membrane protein 1 (VAMP-1 or synaptobrevin 1), whereas astrocytes were stained with an antibody against the glial fibrillary acidic protein (GFAP). Note that VAMP-1-positive nerve terminals (red) are endowed with the P2X7R (green; panel A ), but no significant co-localization was observed between P2X7R (green) and GFAP (red; panel B ); scale bars = 50 μm. Data in panels (C) and (D) correspond to staining overlap and Pearson’s Coefficient (ρ) parameters calculated from three to four confocal micrographs per individual; at least three individuals from each group, control, and MTLE-HS, were analyzed. These parameters were automatically calculated per image with Olympus Fluoview 4.2 Software (Olympus FV1000, Tokyo, Japan) and were used to estimate the co-localization of P2X7R and type-specific cell markers (yellow staining). Overlap between two colors gives values between +1 (total overlap) and 0 (no overlap); the Pearson’s Coefficient (ρ) is a measure of the linear correlation between two variables (stainings), giving values between +1 and −1 inclusive, where 1 is total positive correlation, 0 is no correlation, and −1 is total negative correlation. Higher magnification images show that the P2X7R immunoreactivity also co-localizes with the synaptic vesicle glicoprotein synaptophysin (Synapt), which is one of the most commonly used neuronal cell markers in neuropathology (Panel E ), but not with GFAP (Panel F ). Nuclei are stained with DAPI; cross-reactivity for the secondary antibodies was tested in control experiments in which primary antibodies were omitted (negative controls).
    Figure Legend Snippet: Representative confocal micrographs showing that the P2X7R immunoreactivity is located predominantly in nerve terminals, but not in glial cells, of all sub-regions of the hippocampus of MTLE-HS patients. Synaptic nerve terminals were identified with an antibody against the vesicle-associated membrane protein 1 (VAMP-1 or synaptobrevin 1), whereas astrocytes were stained with an antibody against the glial fibrillary acidic protein (GFAP). Note that VAMP-1-positive nerve terminals (red) are endowed with the P2X7R (green; panel A ), but no significant co-localization was observed between P2X7R (green) and GFAP (red; panel B ); scale bars = 50 μm. Data in panels (C) and (D) correspond to staining overlap and Pearson’s Coefficient (ρ) parameters calculated from three to four confocal micrographs per individual; at least three individuals from each group, control, and MTLE-HS, were analyzed. These parameters were automatically calculated per image with Olympus Fluoview 4.2 Software (Olympus FV1000, Tokyo, Japan) and were used to estimate the co-localization of P2X7R and type-specific cell markers (yellow staining). Overlap between two colors gives values between +1 (total overlap) and 0 (no overlap); the Pearson’s Coefficient (ρ) is a measure of the linear correlation between two variables (stainings), giving values between +1 and −1 inclusive, where 1 is total positive correlation, 0 is no correlation, and −1 is total negative correlation. Higher magnification images show that the P2X7R immunoreactivity also co-localizes with the synaptic vesicle glicoprotein synaptophysin (Synapt), which is one of the most commonly used neuronal cell markers in neuropathology (Panel E ), but not with GFAP (Panel F ). Nuclei are stained with DAPI; cross-reactivity for the secondary antibodies was tested in control experiments in which primary antibodies were omitted (negative controls).

    Techniques Used: Staining, Software

    The inverse relationship between miR-22 serum levels and the P2X7R expression in the hippocampus (A) and temporal neocortex (B) from drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy. Ordinates represent ΔCt variation of P2X7R and miR-22 expression in epileptic patients compared to the corresponding median of non-epileptic controls; 0 represents null variation; positive and negative values correspond to increases and decreases relative to the control population, respectively. Please note that there is a mismatch between miR-22 serum quantifications and P2X7R mRNA determinations in the hippocampus ( A , two patients missing) and temporal neocortex ( B , one patient missing) because RNA samples were insufficient or did not pass the quality assessment.
    Figure Legend Snippet: The inverse relationship between miR-22 serum levels and the P2X7R expression in the hippocampus (A) and temporal neocortex (B) from drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy. Ordinates represent ΔCt variation of P2X7R and miR-22 expression in epileptic patients compared to the corresponding median of non-epileptic controls; 0 represents null variation; positive and negative values correspond to increases and decreases relative to the control population, respectively. Please note that there is a mismatch between miR-22 serum quantifications and P2X7R mRNA determinations in the hippocampus ( A , two patients missing) and temporal neocortex ( B , one patient missing) because RNA samples were insufficient or did not pass the quality assessment.

    Techniques Used: Expressing

    The P2X7R mRNA is overexpressed in the hippocampus and temporal neocortex of drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy compared to non-epileptic cadaveric controls (CTR). qPCR data are expressed as mean ± SD; n numbers inside each bar represent the number of individuals among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. * p
    Figure Legend Snippet: The P2X7R mRNA is overexpressed in the hippocampus and temporal neocortex of drug-refractory MTLE-HS patients submitted to amygdalohippocampectomy compared to non-epileptic cadaveric controls (CTR). qPCR data are expressed as mean ± SD; n numbers inside each bar represent the number of individuals among 10 controls and 23 MTLE-HS patients in which quality assessment of retrieved RNA samples was suitable for quantification. * p

    Techniques Used: Real-time Polymerase Chain Reaction

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    Alomone Labs rabbit polyclonal anti human p2x7 receptor atto 488
    TLR4 signaling influences <t>P2X7</t> receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P
    Rabbit Polyclonal Anti Human P2x7 Receptor Atto 488, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit polyclonal p2x7 c terminus antibody
    The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in  gray , P2X1 aa20–23 and equivalent residues in P2X2  underlined , and the P2X7 residues Lys 17  and Val 18  in  red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7.  C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT:  n  = 14 alone,  n  = 11 MCD,  n  = 3 cholesterol; mutants:  n  = 12 alone,  n  = 11 MCD,  n  = 3 cholesterol; *,  p
    Rabbit Polyclonal P2x7 C Terminus Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti p2x7 receptor antibody
    <t>P2X7</t> receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P
    Anti P2x7 Receptor Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TLR4 signaling influences P2X7 receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways

    doi: 10.3389/fphar.2017.00350

    Figure Lengend Snippet: TLR4 signaling influences P2X7 receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P

    Article Snippet: LMMP whole mount preparations or ileal cryosections were then incubated overnight at room temperature with the following antibodies: chicken polyclonal anti-mouse glial fibrillary acidic protein (GFAP; 1:100; Abcam, Milan, Italy), rabbit polyclonal anti-human GFAP (1:200; Merck Millipore Corporation, Milan, Italy), mouse biotin-conjugated anti-human HuC/D (1:50; Thermo Fisher Scientific, Milan, Italy), rabbit polyclonal anti-mouse neuronal nitric oxide synthase (nNOS, 1:100; Thermo Fisher Scientific, Milan, Italy), rabbit polyclonal anti-human inducible NOS (iNOS; 1:50, Santa Cruz Biotechnology, Milan, Italy), rabbit polyclonal anti-human vasoactive intestinal peptide (VIP, 1:100; GenWay Biotech, Milan, Italy), rabbit polyclonal anti-human P2X7 receptor-ATTO-488 (1:100; Alomone labs, Jerusalem, Israel), rabbit polyclonal anti-human P2Y1 receptor (1:100; Alomone labs, Jerusalem, Israel), rabbit monoclonal anti-human S100β (1:100; Merck Millipore Corporation, Milan, Italy) and guinea pig polyclonal anti-mouse substance P (1:100; Abcam, Milan, Italy).

    Techniques: Fluorescence, Mouse Assay, Staining

    The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in  gray , P2X1 aa20–23 and equivalent residues in P2X2  underlined , and the P2X7 residues Lys 17  and Val 18  in  red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7.  C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT:  n  = 14 alone,  n  = 11 MCD,  n  = 3 cholesterol; mutants:  n  = 12 alone,  n  = 11 MCD,  n  = 3 cholesterol; *,  p

    Journal: The Journal of Biological Chemistry

    Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

    doi: 10.1074/jbc.M114.574699

    Figure Lengend Snippet: The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in gray , P2X1 aa20–23 and equivalent residues in P2X2 underlined , and the P2X7 residues Lys 17 and Val 18 in red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7. C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT: n = 14 alone, n = 11 MCD, n = 3 cholesterol; mutants: n = 12 alone, n = 11 MCD, n = 3 cholesterol; *, p

    Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

    Techniques: Activity Assay, Sequencing, Mutagenesis, Relative Rate

    P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

    Journal: bioRxiv

    Article Title: Asymmetric activation of microglia in the hippocampus drives anxiodepressive consequences of trigeminal neuralgia

    doi: 10.1101/2022.04.16.488241

    Figure Lengend Snippet: P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

    Article Snippet: We used the following primary antibodies: rabbit anti-P2X7R (1:2000, #APR-004, Alomone labs, Jerusalem, Israel), goat-anti IBA-1 (1:1000, #ab5076, Abcam, Cambridge, MA), and rabbit-anti IL-1β (1:500, rabbit, PeproTech, Rocky Hill, NJ).

    Techniques: Activation Assay, Western Blot

    P2X7 inhibition reduced the ATP release with simultaneous decrease of some pro-inflammatory cytokine levels in mouse serum and changed the redox metabolism in C6 tumors. a An ATP release is significantly lower in tumors treated with BBG compared to the control tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Decline NOS-2 expression in BBG-treated tumors ( n = 4). c Decreased expression of FOXP3 (T reg lymphocytes) and CD68 (macrophages) markers in BBG-treated tumors evaluated using flow cytometry ( n = 5). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d The level of the pro-inflammatory and anti-inflammatory cytokines, in mouse serum and glioma tumors using cytokine array for multiplex protein detection ( n = 3). e P2X7 inhibition caused a decrease of GSSG/GSH ratio with a slight decline of SOD activity in BBG-treated tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: P2X7 inhibition reduced the ATP release with simultaneous decrease of some pro-inflammatory cytokine levels in mouse serum and changed the redox metabolism in C6 tumors. a An ATP release is significantly lower in tumors treated with BBG compared to the control tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Decline NOS-2 expression in BBG-treated tumors ( n = 4). c Decreased expression of FOXP3 (T reg lymphocytes) and CD68 (macrophages) markers in BBG-treated tumors evaluated using flow cytometry ( n = 5). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d The level of the pro-inflammatory and anti-inflammatory cytokines, in mouse serum and glioma tumors using cytokine array for multiplex protein detection ( n = 3). e P2X7 inhibition caused a decrease of GSSG/GSH ratio with a slight decline of SOD activity in BBG-treated tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: Inhibition, Expressing, Flow Cytometry, Multiplex Assay, Activity Assay

    P2X7 receptor effects on human glioma development. a Western blot analysis of p-p38 MAPK, CD-133 and HSPA1 expression in U-138 human glioma cells ( n = 3). b In silico analysis of P2X7 expression level and survival of glioma patients using information from the TCGA database. c In silico analysis of P2X7 expression level in different WHO astrocytoma grades compared to normal tissue using NCBI dataset. d A transwell migration assay of LN-229 and U-251 glioma cells treated with 2 μM KN-62 for 24 h ( n = 3). e A crystal violet assay demonstrating a synergistic effect of 2 μM KN-62 with BCNU/TMZ co-treatment on LN-229, U-251, and U-138 glioma cell growth for 24 h ( n = 4). The significance of the differences was determined with repeated-measures one-way ANOVA with Duncan multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: P2X7 receptor effects on human glioma development. a Western blot analysis of p-p38 MAPK, CD-133 and HSPA1 expression in U-138 human glioma cells ( n = 3). b In silico analysis of P2X7 expression level and survival of glioma patients using information from the TCGA database. c In silico analysis of P2X7 expression level in different WHO astrocytoma grades compared to normal tissue using NCBI dataset. d A transwell migration assay of LN-229 and U-251 glioma cells treated with 2 μM KN-62 for 24 h ( n = 3). e A crystal violet assay demonstrating a synergistic effect of 2 μM KN-62 with BCNU/TMZ co-treatment on LN-229, U-251, and U-138 glioma cell growth for 24 h ( n = 4). The significance of the differences was determined with repeated-measures one-way ANOVA with Duncan multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: Western Blot, Expressing, In Silico, Transwell Migration Assay, Crystal Violet Assay

    Characterization of P2X7 receptor in glioma C6 cells in vitro. a Western blot analysis of P2X7 protein expression in C6 cells after P2X7 silencing for 72 h ( n = 3). b Calcium signal level in glioma C6 cells after P2X7 downregulation and BzATP stimulation (300 µM) ( n = 4). Gray lines represent the signals from the biological replicates; black lines represent the signal averaged among the individual replicates. Insert shows effect of P2X7 downregulation on the signal strength, one-way t -test: * P ≤ 0.05. c Measurement of bioluminescence originated from released ATP after P2X7 activation/inhibition in C6 cells ( n = 3). The statistical significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: Characterization of P2X7 receptor in glioma C6 cells in vitro. a Western blot analysis of P2X7 protein expression in C6 cells after P2X7 silencing for 72 h ( n = 3). b Calcium signal level in glioma C6 cells after P2X7 downregulation and BzATP stimulation (300 µM) ( n = 4). Gray lines represent the signals from the biological replicates; black lines represent the signal averaged among the individual replicates. Insert shows effect of P2X7 downregulation on the signal strength, one-way t -test: * P ≤ 0.05. c Measurement of bioluminescence originated from released ATP after P2X7 activation/inhibition in C6 cells ( n = 3). The statistical significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Inhibition

    P2X7 activation increased C6 cell proliferation, viability, and cell adhesion in vitro. Quantitative data are presented as signal relative to control. a MTS test of long-term C6 cell viability after 100 µM BzATP stimulation of control cells and cells with P2X7 downregulation using RNAi ( n = 3). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot analysis of pro-survival and pro-proliferative proteins: p-p38 (Thr180/Tyr182), pAkt (Ser473), tAkt, CD133, HSPA1, and HSPA5 in C6 cell lysates after 24-h BzATP stimulation ( n = 3). c Upper panel: MTS test of C6 cell viability after 200 µM BCNU (carmustine) treatment for 24 h. Co-treatment with 100 µM BzATP and 200 µM BCNU increased viability of C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. Bottom panel: a crystal violet assay demonstrating a synergistic effect of BBG with BCNU/TMZ co-treatment on C6 cell growth for 24 h ( n = 6). The significance of the differences was determined with repeated measures one-way ANOVA with Duncan’s multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. d P2X7 activation affects C6 cell adhesion to extracellular matrix components: collagen I, collagen IV, fibronectin. C6 cells showed higher adhesion potential after 24-h BzATP stimulation. 24-h co-treatment/treatment with 100 nM BBG significantly decreased C6 cell adhesion to ECM components ( n = 6). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. e P2X7 RNAi downregulation declined C6 cell adhesion to ECM components upon BzATP stimulation ( n = 4). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: P2X7 activation increased C6 cell proliferation, viability, and cell adhesion in vitro. Quantitative data are presented as signal relative to control. a MTS test of long-term C6 cell viability after 100 µM BzATP stimulation of control cells and cells with P2X7 downregulation using RNAi ( n = 3). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot analysis of pro-survival and pro-proliferative proteins: p-p38 (Thr180/Tyr182), pAkt (Ser473), tAkt, CD133, HSPA1, and HSPA5 in C6 cell lysates after 24-h BzATP stimulation ( n = 3). c Upper panel: MTS test of C6 cell viability after 200 µM BCNU (carmustine) treatment for 24 h. Co-treatment with 100 µM BzATP and 200 µM BCNU increased viability of C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. Bottom panel: a crystal violet assay demonstrating a synergistic effect of BBG with BCNU/TMZ co-treatment on C6 cell growth for 24 h ( n = 6). The significance of the differences was determined with repeated measures one-way ANOVA with Duncan’s multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. d P2X7 activation affects C6 cell adhesion to extracellular matrix components: collagen I, collagen IV, fibronectin. C6 cells showed higher adhesion potential after 24-h BzATP stimulation. 24-h co-treatment/treatment with 100 nM BBG significantly decreased C6 cell adhesion to ECM components ( n = 6). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. e P2X7 RNAi downregulation declined C6 cell adhesion to ECM components upon BzATP stimulation ( n = 4). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: Activation Assay, In Vitro, Western Blot, Crystal Violet Assay

    Effect of P2X7 inhibition using BBG on glioma C6 tumor development in vivo. a Brilliant Blue G administration led to reduction of glioma tumor mass. Representative images of control and BBG-treated C6 glioma tumors ( n = 9 for control group and n = 10 for BBG-treated group). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot showing the significant decrease of P2X7 expression in tumor homogenates after BBG treatment. Representative image of P2X7 immunodetection in C6 glioma tumors. c P2X7 inhibition resulted in diminished activation of matrix metalloproteinase-2 in glioma tumors. The significance of the differences was determined with Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d Western blot analysis of known proteins involved in cell adhesion and EMT signaling in C6 tumor homogenates ( n = 4). e Western blot analysis of known proteins involved in tumor aggressiveness in C6 tumor homogenates ( n = 4)

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: Effect of P2X7 inhibition using BBG on glioma C6 tumor development in vivo. a Brilliant Blue G administration led to reduction of glioma tumor mass. Representative images of control and BBG-treated C6 glioma tumors ( n = 9 for control group and n = 10 for BBG-treated group). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot showing the significant decrease of P2X7 expression in tumor homogenates after BBG treatment. Representative image of P2X7 immunodetection in C6 glioma tumors. c P2X7 inhibition resulted in diminished activation of matrix metalloproteinase-2 in glioma tumors. The significance of the differences was determined with Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d Western blot analysis of known proteins involved in cell adhesion and EMT signaling in C6 tumor homogenates ( n = 4). e Western blot analysis of known proteins involved in tumor aggressiveness in C6 tumor homogenates ( n = 4)

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: Inhibition, In Vivo, Western Blot, Expressing, Immunodetection, Activation Assay

    Activation of P2X7 receptor led to elevated ROS production and mitochondrial membrane depolarization in C6 cells. Quantitative data are presented as signal relative to control. a DCF-DA fluorescence measurement in C6 glioma cells. Cells stimulated with 100 µM BzATP for 1 h were characterized by increased ROS production compared to control in C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. b Potential-dependent staining of mitochondria in C6 cells using JC-1. 1-h BzATP (100 µM) stimulation led to considerable mitochondrial membrane depolarization ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. c Representative images of MitoTracker Red CMXRos accumulation in C6 cells stimulated with 100 µM BzATP for 1 h. d DCF-DA fluorescence measurement in C6 glioma cells with RNA interference of P2X7 expression ( n = 4). Downregulation of P2X7 led to decreased ROS production in C6 cells after 1-h BzATP (100 µM) stimulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. e Potential-dependent staining of mitochondria using JC-1 dye in C6 cells with RNA interference of P2X7 expression after 1 h of 100 μM BzATP pre-treatment ( n = 3). The amount of depolarized mitochondria was higher in control cells when compared to that in cells with P2X7 downregulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Journal: Purinergic Signalling

    Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

    doi: 10.1007/s11302-021-09834-2

    Figure Lengend Snippet: Activation of P2X7 receptor led to elevated ROS production and mitochondrial membrane depolarization in C6 cells. Quantitative data are presented as signal relative to control. a DCF-DA fluorescence measurement in C6 glioma cells. Cells stimulated with 100 µM BzATP for 1 h were characterized by increased ROS production compared to control in C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. b Potential-dependent staining of mitochondria in C6 cells using JC-1. 1-h BzATP (100 µM) stimulation led to considerable mitochondrial membrane depolarization ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. c Representative images of MitoTracker Red CMXRos accumulation in C6 cells stimulated with 100 µM BzATP for 1 h. d DCF-DA fluorescence measurement in C6 glioma cells with RNA interference of P2X7 expression ( n = 4). Downregulation of P2X7 led to decreased ROS production in C6 cells after 1-h BzATP (100 µM) stimulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. e Potential-dependent staining of mitochondria using JC-1 dye in C6 cells with RNA interference of P2X7 expression after 1 h of 100 μM BzATP pre-treatment ( n = 3). The amount of depolarized mitochondria was higher in control cells when compared to that in cells with P2X7 downregulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

    Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

    Techniques: Activation Assay, Fluorescence, Staining, Expressing