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anti rabbit p2y 6 r polyclonal antibody (Alomone Labs)

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Alomone Labs anti rabbit p2y 6 r polyclonal antibody

Endothelial <t>P2Y</t> <t>6</t> <t>R</t> activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

Anti Rabbit P2y 6 R Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+p2y/Anti-LPAR4+Antibody/pmc13013911-54-19-26
Average 90 stars, based on 4 article reviews

anti rabbit p2y 6 r polyclonal antibody - by Bioz Stars, 2026-09

90/100 stars

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1) Product Images from "Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis"

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

Journal: Purinergic Signalling

doi: 10.1007/s11302-026-10141-x

Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

Figure Legend Snippet: Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

Techniques Used: Activation Assay, Control, Infection

Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

Figure Legend Snippet: Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

Techniques Used: Immunocytochemistry, Staining, Cell Culture, Control, Infection, Fluorescence

Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

Figure Legend Snippet: Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

Techniques Used:

Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

Figure Legend Snippet: Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

Techniques Used: Expressing, Inhibition, Immunocytochemistry, Staining, Cell Culture, Fluorescence

Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

Figure Legend Snippet: Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

Techniques Used: Infection, Expressing

Incubation:

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling
Article Snippet: .. Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA). .. Cells were then washed and incubated at room temperature for 1 h with the secondary antibody (diluted to 1:300, in 0.1% BSA in PBS): anti-rabbit IgG (H+L)-Alexa Fluor ® 594 (Thermo Fisher Scientific, NJ, USA).

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Journal: Purinergic Signalling

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

doi: 10.1007/s11302-026-10141-x

Figure Lengend Snippet: Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

Techniques: Activation Assay, Control, Infection

Journal: Purinergic Signalling

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

doi: 10.1007/s11302-026-10141-x

Figure Lengend Snippet: Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Infection, Fluorescence

Journal: Purinergic Signalling

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

doi: 10.1007/s11302-026-10141-x

Figure Lengend Snippet: Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

Techniques:

Journal: Purinergic Signalling

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

doi: 10.1007/s11302-026-10141-x

Figure Lengend Snippet: Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

Techniques: Expressing, Inhibition, Immunocytochemistry, Staining, Cell Culture, Fluorescence

Journal: Purinergic Signalling

Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

doi: 10.1007/s11302-026-10141-x

Figure Lengend Snippet: Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

Techniques: Infection, Expressing

P2Y 11 and IL-1R cooperate to upregulate CXCR7 surface expression and CCL20 production in primary human M2 macrophages: potentiation by PDE4 inhibition. a M2 macrophages were treated for 24 h with ATPγS (20 µM) alone or in combination with IL-1ß (2 ng/ml) in the presence or absence of rolipram (10 µM). Flow cytometry was used to determine CXCR7 expression (light blue; isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b P2Y 11 receptor antagonist NF340 (20 µM) served to confirm that ATPγS-mediated changes were specific to P2Y 11 receptor activation. c Quantification of CXCR7 expression ( n = 3): mean values ± SD are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001. D) Quantification of CCL20 production after the same treatments that were used to upregulate CXCR7 expression ( n = 5). Mean values ± SD are shown. **** p < 0.0001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: P2Y 11 and IL-1R cooperate to upregulate CXCR7 surface expression and CCL20 production in primary human M2 macrophages: potentiation by PDE4 inhibition. a M2 macrophages were treated for 24 h with ATPγS (20 µM) alone or in combination with IL-1ß (2 ng/ml) in the presence or absence of rolipram (10 µM). Flow cytometry was used to determine CXCR7 expression (light blue; isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b P2Y 11 receptor antagonist NF340 (20 µM) served to confirm that ATPγS-mediated changes were specific to P2Y 11 receptor activation. c Quantification of CXCR7 expression ( n = 3): mean values ± SD are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001. D) Quantification of CCL20 production after the same treatments that were used to upregulate CXCR7 expression ( n = 5). Mean values ± SD are shown. **** p < 0.0001

Article Snippet: The following antibodies were used: rabbit polyclonal IgG anti-human P2Y 11 receptor (bs-12071R-A-488; Bioss, Woburn, MA, USA), mouse monoclonal IgG 1 anti-human P2Y 11 /P2RY11 (FAB9305R-025-AF647; R&D Systems), mouse monoclonal IgG2b anti-human CD14 (clone MϕP9, 345,787-APC; BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal IgG1,k anti-human CD163 (clone GHI/61, 556,018-PE; BD Biosciences), mouse monoclonal IgG 1 ,k anti-human CD206 (clone 19.2, 17-2069-42-APC, Invitrogen / Thermo Fisher Scientific), mouse monoclonal IgG2a,k anti-human CD184/CXCR4 (clone 12G5, 555,974-PE; BD Biosciences), mouse monoclonal IgG 1 anti-human ACKR3/CXCR7 (LS-C128442-APC; Lifespan Biosciences, Lynnwood, WA, USA), and goat polyclonal IgG anti-human IL-1R1 (FAB269P-PE; R&D Systems).

Techniques: Expressing, Inhibition, Flow Cytometry, Fluorescence, Control, Activation Assay

P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation in primary human M2 macrophages is mediated by EGFR. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß to induce CXCR7 upregulation. (B) Potentiation of CXCR7 expression by PDE4 inhibitor rolipram. a,b To examine EGFR involvement, EGFR-TKIs AG1478 and erlotinib were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation. Representative FACS histograms of CXCR7 expression (light blue; isotype controls in red) are shown in the left panel. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. a,b Quantification of CXCR7 expression ( n = 3) and CCL20 production ( n = 5) is shown in the right panel. Data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation in primary human M2 macrophages is mediated by EGFR. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß to induce CXCR7 upregulation. (B) Potentiation of CXCR7 expression by PDE4 inhibitor rolipram. a,b To examine EGFR involvement, EGFR-TKIs AG1478 and erlotinib were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation. Representative FACS histograms of CXCR7 expression (light blue; isotype controls in red) are shown in the left panel. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. a,b Quantification of CXCR7 expression ( n = 3) and CCL20 production ( n = 5) is shown in the right panel. Data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Techniques: Expressing, Fluorescence, Staining, Control

P2Y 11 activation is sufficient to induce high CXCR4 expression in primary human M2 macrophages: enhancement by PDE4 inhibition with no requirement for IL-1ß co-stimulation. a M2 macrophages were treated for 6 h with P2Y 11 receptor agonist ATPγS ± PDE4 inhibitor rolipram. Copy numbers of CXCR4 mRNA were determined using NanoString technology. P2Y 11 receptor antagonist NF340 was used to confirm that ATPγS-mediated changes were specific to P2Y 11 receptor activation. *** p < 0.001, **** p < 0.0001. b M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS ± PDE4 inhibitor rolipram. Flow cytometry was used to determine CXCR4 expression (light blue; isotype controls in red). Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. c Quantification of CXCR4 expression ( n = 5). Mean values ± SD are shown. * p < 0.05, ** p < 0.01, *** p < 0.001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: P2Y 11 activation is sufficient to induce high CXCR4 expression in primary human M2 macrophages: enhancement by PDE4 inhibition with no requirement for IL-1ß co-stimulation. a M2 macrophages were treated for 6 h with P2Y 11 receptor agonist ATPγS ± PDE4 inhibitor rolipram. Copy numbers of CXCR4 mRNA were determined using NanoString technology. P2Y 11 receptor antagonist NF340 was used to confirm that ATPγS-mediated changes were specific to P2Y 11 receptor activation. *** p < 0.001, **** p < 0.0001. b M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS ± PDE4 inhibitor rolipram. Flow cytometry was used to determine CXCR4 expression (light blue; isotype controls in red). Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. c Quantification of CXCR4 expression ( n = 5). Mean values ± SD are shown. * p < 0.05, ** p < 0.01, *** p < 0.001

Techniques: Activation Assay, Expressing, Inhibition, Flow Cytometry, Fluorescence, Staining, Control

CXCR4 antagonists cause CXCR4 downregulation in primary human M2 macrophages. M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the a absence or b presence of PDE4 inhibitor rolipram. a,b The CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR4 expression. Representative FACS histograms of CXCR4 expression (light blue; isotype controls in red) are shown in the left panel. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. a,b Quantification of CXCR4 expression is shown in the right panel ( n = 4). Mean values ± SD are shown. *** p < 0.001, **** p < 0.0001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: CXCR4 antagonists cause CXCR4 downregulation in primary human M2 macrophages. M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the a absence or b presence of PDE4 inhibitor rolipram. a,b The CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR4 expression. Representative FACS histograms of CXCR4 expression (light blue; isotype controls in red) are shown in the left panel. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. a,b Quantification of CXCR4 expression is shown in the right panel ( n = 4). Mean values ± SD are shown. *** p < 0.001, **** p < 0.0001

Techniques: Expressing, Fluorescence, Staining, Control

CXCR4 antagonists do not downregulate but rather enhance CXCR7 expression and CCL20 production in primary human M2 macrophages. a CXCR7 expression: M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence (upper panel) or presence of PDE4 inhibitor rolipram (lower panel). CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 expression. Flow cytometry was used to determine CXCR7 expression ( n = 3; mean fluorescence intensities, MFIs, after subtraction of isotype control MFIs). Mean values ± SD are shown. * p < 0.05. b CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced (upper panels; n = 3) and rolipram-enhanced CCL20 production (lower panels; n = 3): data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: CXCR4 antagonists do not downregulate but rather enhance CXCR7 expression and CCL20 production in primary human M2 macrophages. a CXCR7 expression: M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence (upper panel) or presence of PDE4 inhibitor rolipram (lower panel). CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 expression. Flow cytometry was used to determine CXCR7 expression ( n = 3; mean fluorescence intensities, MFIs, after subtraction of isotype control MFIs). Mean values ± SD are shown. * p < 0.05. b CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced (upper panels; n = 3) and rolipram-enhanced CCL20 production (lower panels; n = 3): data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

The P2Y 11 -recombinant astrocytoma cell line naturally expresses CXCR7 but lacks CXCR4: enhancement of CCL20 production through CXCR7 activation and no need for PDE4 inhibition. a Flow cytometry was used to determine P2Y 11 receptor as well as CXCR7 and CXCR4 expression (light blue; the respective isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 5). Mean values ± SD are shown. **** p < 0.0001. c Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 3). Mean values ± SD are shown. * p < 0.05, **** p < 0.0001. d CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced CCL20 production ( n = 3). Data shown are mean values ± SD. ** p < 0.01, **** p < 0.0001

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: The P2Y 11 -recombinant astrocytoma cell line naturally expresses CXCR7 but lacks CXCR4: enhancement of CCL20 production through CXCR7 activation and no need for PDE4 inhibition. a Flow cytometry was used to determine P2Y 11 receptor as well as CXCR7 and CXCR4 expression (light blue; the respective isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 5). Mean values ± SD are shown. **** p < 0.0001. c Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 3). Mean values ± SD are shown. * p < 0.05, **** p < 0.0001. d CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced CCL20 production ( n = 3). Data shown are mean values ± SD. ** p < 0.01, **** p < 0.0001

Techniques: Recombinant, Activation Assay, Inhibition, Flow Cytometry, Expressing, Fluorescence, Control

CXCR7 knockdown by RNA interference in astrocytoma cells attenuates CCL20 expression. a Astrocytoma cells were transfected with either control siRNA or CXCR7 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring CXCR7 surface expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Quantification of CXCR7 knockdown ( n = 4): mean values ± SD are shown. *** p < 0.001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to CXCR7 knockdown (CXCR7 siRNA; n = 5): mean values ± SD are shown. **** p < 0.0001. c Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown. d Astrocytoma cells were transfected with either control siRNA or IL1R1 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring IL-1R1 expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. e Quantification of IL1-R1 knockdown ( n = 4): mean values ± SD are shown. **** p < 0.0001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to IL1R1 knockdown (IL1R1 siRNA; n = 3): mean values ± SD are shown. **** p < 0.0001. f Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: CXCR7 knockdown by RNA interference in astrocytoma cells attenuates CCL20 expression. a Astrocytoma cells were transfected with either control siRNA or CXCR7 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring CXCR7 surface expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Quantification of CXCR7 knockdown ( n = 4): mean values ± SD are shown. *** p < 0.001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to CXCR7 knockdown (CXCR7 siRNA; n = 5): mean values ± SD are shown. **** p < 0.0001. c Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown. d Astrocytoma cells were transfected with either control siRNA or IL1R1 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring IL-1R1 expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. e Quantification of IL1-R1 knockdown ( n = 4): mean values ± SD are shown. **** p < 0.0001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to IL1R1 knockdown (IL1R1 siRNA; n = 3): mean values ± SD are shown. **** p < 0.0001. f Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown

Techniques: Knockdown, Expressing, Transfection, Control, Flow Cytometry, Fluorescence

Synergistic cooperation between P2Y 11 and CXCR7 in primary human macrophages eliminates the need for IL-1ß co-stimulation and PDE4 inhibition. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS and CXCR7 agonist TC14012, alone or in combination, and CCL20 was determined in culture supernatants ( n = 5). Mean values ± SD are shown. **** p < 0.0001. b Flow cytometry was used to determine CXCR7 and CXCR4 expression (light blue; isotype controls in red). Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. c Quantification of CXCR7 ( n = 5) and CXCR4 expression ( n = 3). Mean values ± SD are shown. *** p < 0.001, **** p < 0.0001. d M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence or absence of either CXCR4 antagonist plerixafor or CXCR7 agonist TC14012. Pooled supernatants were analyzed for the presence of 20 CCL chemokines using RayBio technology. Plerixafor- or TC14012-mediated modulation of CCL chemokines that were present in the secretome and produced in a P2Y 11 receptor dependent manner (i.e. sensitive to inhibition with NF340) is shown (see also Fig. )

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: Synergistic cooperation between P2Y 11 and CXCR7 in primary human macrophages eliminates the need for IL-1ß co-stimulation and PDE4 inhibition. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS and CXCR7 agonist TC14012, alone or in combination, and CCL20 was determined in culture supernatants ( n = 5). Mean values ± SD are shown. **** p < 0.0001. b Flow cytometry was used to determine CXCR7 and CXCR4 expression (light blue; isotype controls in red). Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. c Quantification of CXCR7 ( n = 5) and CXCR4 expression ( n = 3). Mean values ± SD are shown. *** p < 0.001, **** p < 0.0001. d M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence or absence of either CXCR4 antagonist plerixafor or CXCR7 agonist TC14012. Pooled supernatants were analyzed for the presence of 20 CCL chemokines using RayBio technology. Plerixafor- or TC14012-mediated modulation of CCL chemokines that were present in the secretome and produced in a P2Y 11 receptor dependent manner (i.e. sensitive to inhibition with NF340) is shown (see also Fig. )

Techniques: Inhibition, Flow Cytometry, Expressing, Fluorescence, Staining, Control, Produced

Working model of P2Y 11 crosstalk with IL-1R and chemokine receptors summarizing current findings and knowledge. P2Y 11 couples to G q and G s proteins. While G q activates phospholipase Cß and thus initiates the mobilization of Ca 2+ (via inositol triphosphate, IP 3 ) as well as the activation of protein kinase C (PKC, via diacylglycerol, DAG), G s activates adenylyl cyclase (AC) to increase the levels of cyclic adenosine monophosphate (cyclic AMP). P2Y 11 upregulates CXCR4 and via IL-1R also CXCR7. Upregulation of IL-1R, CXCR7 and CCL20 depends on cyclic AMP. Epidermal growth factor receptor (EGFR) supports upregulation of CXCR7, which serves as an EGFR activator potentiating its signaling capacity, generating a feed-forward loop. Thus, CXCR7 stimulates its own expression with the help of EGFR. By coupling to G i , which blocks the cyclic AMP-generating enzyme AC, and by recruiting the cyclic AMP-degrading enzyme PDE4 via ß-arrestin, CXCR4 serves as a perfectly suited sentinel of intracellular cyclic AMP levels and thus as a regulatory checkpoint of P2Y 11 /IL-1R induced and EGFR/CXCR7-mediated responses (dashed inhibitory arrow) such as the selective activation of CCL20 secretion. Once CXCR7 is fully activated by TC14012, it takes control and eliminates CXCR4 expression (inhibitory arrow) and thus also the need for rolipram-mediated PDE4 inhibition

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: Working model of P2Y 11 crosstalk with IL-1R and chemokine receptors summarizing current findings and knowledge. P2Y 11 couples to G q and G s proteins. While G q activates phospholipase Cß and thus initiates the mobilization of Ca 2+ (via inositol triphosphate, IP 3 ) as well as the activation of protein kinase C (PKC, via diacylglycerol, DAG), G s activates adenylyl cyclase (AC) to increase the levels of cyclic adenosine monophosphate (cyclic AMP). P2Y 11 upregulates CXCR4 and via IL-1R also CXCR7. Upregulation of IL-1R, CXCR7 and CCL20 depends on cyclic AMP. Epidermal growth factor receptor (EGFR) supports upregulation of CXCR7, which serves as an EGFR activator potentiating its signaling capacity, generating a feed-forward loop. Thus, CXCR7 stimulates its own expression with the help of EGFR. By coupling to G i , which blocks the cyclic AMP-generating enzyme AC, and by recruiting the cyclic AMP-degrading enzyme PDE4 via ß-arrestin, CXCR4 serves as a perfectly suited sentinel of intracellular cyclic AMP levels and thus as a regulatory checkpoint of P2Y 11 /IL-1R induced and EGFR/CXCR7-mediated responses (dashed inhibitory arrow) such as the selective activation of CCL20 secretion. Once CXCR7 is fully activated by TC14012, it takes control and eliminates CXCR4 expression (inhibitory arrow) and thus also the need for rolipram-mediated PDE4 inhibition

Techniques: Activation Assay, Expressing, Control, Inhibition

$The P2Y 13 receptor is expressed by qNSCs in the adult SEZ. A. The expression of the different metabotropic P2Y receptors in the adult SEZ was analyzed by quantitative RT-PCR (n=6). B. P2Y 13 receptor expression in the ventral and dorsal wall of the adult SEZ analyzed by quantitative RT-PCR (n=6). C . Comparison of the P2Y 13 receptor protein in the ventral and dorsal wall of the SEZ (n=3). D-F . P2Y 13 receptor expression in the ventral wall of the SEZ. Note how the P2Y 13 receptor (red) co-localizes with GFAP positive (white) astroglia (yellow arrowheads) but not with Ascl1 positive TAPs or cells expressing βIII-tubulin (green, scale bar 30 µm except for Ascl1 10 µm). G . Co-localization of the P2Y 13 receptor (red) in NSCs with SOX2 (white) and GFAP (green) in the adult SEZ (scale bar 30 µm). H. SEZ-derived cell populations expressing the P2Y 13 receptor in a FACs analysis using a P2Y 13 -GFP conjugated antibody. Note how the expression is mainly associated with the qNSC fraction (n=3). All graphs show the mean ±SEM: *p<0.05, **p<0.01, and ***p<0.001 (T-test).$

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is expressed by qNSCs in the adult SEZ. A. The expression of the different metabotropic P2Y receptors in the adult SEZ was analyzed by quantitative RT-PCR (n=6). B. P2Y 13 receptor expression in the ventral and dorsal wall of the adult SEZ analyzed by quantitative RT-PCR (n=6). C . Comparison of the P2Y 13 receptor protein in the ventral and dorsal wall of the SEZ (n=3). D-F . P2Y 13 receptor expression in the ventral wall of the SEZ. Note how the P2Y 13 receptor (red) co-localizes with GFAP positive (white) astroglia (yellow arrowheads) but not with Ascl1 positive TAPs or cells expressing βIII-tubulin (green, scale bar 30 µm except for Ascl1 10 µm). G . Co-localization of the P2Y 13 receptor (red) in NSCs with SOX2 (white) and GFAP (green) in the adult SEZ (scale bar 30 µm). H. SEZ-derived cell populations expressing the P2Y 13 receptor in a FACs analysis using a P2Y 13 -GFP conjugated antibody. Note how the expression is mainly associated with the qNSC fraction (n=3). All graphs show the mean ±SEM: *p<0.05, **p<0.01, and ***p<0.001 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Derivative Assay

Expression of the P2Y 13 receptor in the neurogenic lineage of the SEZ-derived cell cultures after 6 DIV: GFAP (magenta), Ascl1 (green, middle panel), βIII-tubulin (green, lower panel), P2Y 13 (red). Note how P2Y 13 receptor expression only co-localizes with GFAP in cells. Scale bar 30 µm.

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Expression of the P2Y 13 receptor in the neurogenic lineage of the SEZ-derived cell cultures after 6 DIV: GFAP (magenta), Ascl1 (green, middle panel), βIII-tubulin (green, lower panel), P2Y 13 (red). Note how P2Y 13 receptor expression only co-localizes with GFAP in cells. Scale bar 30 µm.

Techniques: Expressing, Derivative Assay

The P2Y 13 receptor is active in SEZ-derived NSCs in culture. NSCs in culture and loaded with the calcium dye Fura-2 were stimulated with 2MeSADP and after a washout period, they were re-exposed to 2MeSADP in the presence of either the P2Y1 receptor antagonist MRS2179 A or the P2Y 13 receptor antagonist MRS2179 B . All compounds were tested at a concentration of 10 μM and representative traces of the F340/F380 fluorescence ratios recorded from single cells are shown. C . Immunocytochemistry identifying GFAP (green) and SOX2 (red) double positive NSCs that respond to the selective agonist of the P2Y 13 receptor, 2MeSADP (yellow arrowheads). Conversely, the intracellular calcium levels of GFAP positive parenchymal astrocytes (white arrow) does not change. D . Effects of 2MeSADP on voltage-gated currents in SEZ-derived NSCs in culture. Outward currents evoked by a depolarising pulse (+10 mV, 100 ms from a Vh of −80 mV) were increased in the presence of 2MeSADP (10 µM, 2 min). Subsequent exposure to MRS221 (10 µM, 2 min) partially reversed the potentiating effect of 2MeSADP (upper panel). At variance, exposure to MRS2179 (10 µM, 2 min) of 2MeSADP with MRS2179 (10 µM, 2 min) did not have any effect on outward current increase elicited by 2MeSADP (10 µM, 2 min) (lower panel). E . Scatter plot of outward current charges from the experiments shown in ( D ), in the presence or absence of 2MeSADP (10 µM, 2 min: left panel), and following co-incubation (right panels) with MRS2211 (upper right) or MRS2179 (lower right). The values are the means ± SEM of the number of cells indicated between parentheses; the statistical significance was assessed using the student’s T-test for paired samples: *p < 0.05; **p < 0.01.

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is active in SEZ-derived NSCs in culture. NSCs in culture and loaded with the calcium dye Fura-2 were stimulated with 2MeSADP and after a washout period, they were re-exposed to 2MeSADP in the presence of either the P2Y1 receptor antagonist MRS2179 A or the P2Y 13 receptor antagonist MRS2179 B . All compounds were tested at a concentration of 10 μM and representative traces of the F340/F380 fluorescence ratios recorded from single cells are shown. C . Immunocytochemistry identifying GFAP (green) and SOX2 (red) double positive NSCs that respond to the selective agonist of the P2Y 13 receptor, 2MeSADP (yellow arrowheads). Conversely, the intracellular calcium levels of GFAP positive parenchymal astrocytes (white arrow) does not change. D . Effects of 2MeSADP on voltage-gated currents in SEZ-derived NSCs in culture. Outward currents evoked by a depolarising pulse (+10 mV, 100 ms from a Vh of −80 mV) were increased in the presence of 2MeSADP (10 µM, 2 min). Subsequent exposure to MRS221 (10 µM, 2 min) partially reversed the potentiating effect of 2MeSADP (upper panel). At variance, exposure to MRS2179 (10 µM, 2 min) of 2MeSADP with MRS2179 (10 µM, 2 min) did not have any effect on outward current increase elicited by 2MeSADP (10 µM, 2 min) (lower panel). E . Scatter plot of outward current charges from the experiments shown in ( D ), in the presence or absence of 2MeSADP (10 µM, 2 min: left panel), and following co-incubation (right panels) with MRS2211 (upper right) or MRS2179 (lower right). The values are the means ± SEM of the number of cells indicated between parentheses; the statistical significance was assessed using the student’s T-test for paired samples: *p < 0.05; **p < 0.01.

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Immunocytochemistry, Incubation

P2Y 13 silencing increases the number of NSCs remaining in the SEZ without promoting proliferation. A. The effect of local P2Y 13 receptor silencing on the NSC population, achieved using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors: Cas9 (Green), SOX2 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). B. Quantification of the Cas9 + /GFAP + /SOX2 + cells remaining in the adult SEZ (n=4). C Effect of the local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the proliferation within the SEZ: Cas9 (Green), Ki67 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). D . Quantification of the Cas9 + /Ki67 + cells SEZ (n=4). E . Effect of local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the neuroblast population: Cas9 (Green), DCX (White) and the cell nuclei are stained with DAPI. The lower panels show the higher magnification of the selected areas (scale bar 50 µm). F. Quantification of the Cas9 + /DCX + cells (n=4). All graphs show mean ±SEM: ***p<0.001 (T-test).

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: P2Y 13 silencing increases the number of NSCs remaining in the SEZ without promoting proliferation. A. The effect of local P2Y 13 receptor silencing on the NSC population, achieved using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors: Cas9 (Green), SOX2 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). B. Quantification of the Cas9 + /GFAP + /SOX2 + cells remaining in the adult SEZ (n=4). C Effect of the local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the proliferation within the SEZ: Cas9 (Green), Ki67 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). D . Quantification of the Cas9 + /Ki67 + cells SEZ (n=4). E . Effect of local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the neuroblast population: Cas9 (Green), DCX (White) and the cell nuclei are stained with DAPI. The lower panels show the higher magnification of the selected areas (scale bar 50 µm). F. Quantification of the Cas9 + /DCX + cells (n=4). All graphs show mean ±SEM: ***p<0.001 (T-test).

Techniques: CRISPR, Staining

Scheme of the strategy to generate lentiviral vectors for the local overexpression of the P2Y 13 receptor. B . Experimental design of lentiviral injection for local overexpression or silencing of the P2Y 13 receptor. C . Positive control of the local overexpression of P2Y 13 receptor. Lentiviral injection in the adult SEZ demonstrated that all LV-GFP-P2Y 13 transduced cells (Green) co-localized with P2Y 13 expression (red).

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Scheme of the strategy to generate lentiviral vectors for the local overexpression of the P2Y 13 receptor. B . Experimental design of lentiviral injection for local overexpression or silencing of the P2Y 13 receptor. C . Positive control of the local overexpression of P2Y 13 receptor. Lentiviral injection in the adult SEZ demonstrated that all LV-GFP-P2Y 13 transduced cells (Green) co-localized with P2Y 13 expression (red).

Techniques: Over Expression, Injection, Positive Control, Expressing

Local overexpression of P2Y 13 receptors augments the proportion of cells in the RMS and decreases the number of NSCs remaining in the ventral wall of the SEZ. A. Effect of P2Y 13 on SEZ dynamics, with local overexpression promoting more cells entering the RMS and less cells remaining in the SEZ (Scale bar 50 µm). B Quantification of GFP positive cells in the ventral wall of the SEZ and RMS following LV-GFP injection (n=4). C Quantification of GFP + cells in the ventral wall of the SEZ and RMS following LV-P2Y 13 -GFP injections (n=4, scale bar 50 µm). D-E. Effect of local overexpression on the remaining GFAP + (white)/SOX2 + (red)/GFP + (Green) cells in the SEZ after LV-GFP or LV-P2Y 13 -GFP injections (n=4, scale bar 50 µM). F. Quantification of GFAP + /SOX2 + /GFP + cells in the ventral wall of the SEZ following LV-GFP or LV-P2Y 13 -GFP injections (n=4). All graphs show the mean ±SEM: *p<0.05 (T-test).

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Local overexpression of P2Y 13 receptors augments the proportion of cells in the RMS and decreases the number of NSCs remaining in the ventral wall of the SEZ. A. Effect of P2Y 13 on SEZ dynamics, with local overexpression promoting more cells entering the RMS and less cells remaining in the SEZ (Scale bar 50 µm). B Quantification of GFP positive cells in the ventral wall of the SEZ and RMS following LV-GFP injection (n=4). C Quantification of GFP + cells in the ventral wall of the SEZ and RMS following LV-P2Y 13 -GFP injections (n=4, scale bar 50 µm). D-E. Effect of local overexpression on the remaining GFAP + (white)/SOX2 + (red)/GFP + (Green) cells in the SEZ after LV-GFP or LV-P2Y 13 -GFP injections (n=4, scale bar 50 µM). F. Quantification of GFAP + /SOX2 + /GFP + cells in the ventral wall of the SEZ following LV-GFP or LV-P2Y 13 -GFP injections (n=4). All graphs show the mean ±SEM: *p<0.05 (T-test).

Techniques: Over Expression, Injection

Transcriptomic analysis of the RNA-seq data obtained from P2Y 13 overexpressing cells in vivo . A. Scheme of the experimental design: right panel created with BioRender.com. B. A principal component analysis (PCA) of the most variably expressed (top 3,000) genes of the samples profiled, colored by condition: control ( FO_C , blue), P2Y 13 overexpression ( FO_Y , green). C. Heat-map of the top 1,000 most variably expressed genes across the samples profiled. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. D. Heat-map of the genes associated with the activation/quiescence equilibrium and self-renewal. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. E. Volcano plot showing the differential expression of genes between P2Y 13 overexpressing and control samples (n=3, PY 13 and 3 controls). Differentially expressed genes (DEGs, adjusted p-value <0.05) are in red and green when upregulated or downregulated in the P2Y 13 overexpressing samples, respectively. The bar plot represents the number of DEGs. F. Overrepresentation of gene ontology (GO) terms for biological processes from the DEGs (adjusted p-value <0.05) between the P2Y 13 overexpressing and control samples. A customized selection (18) of significant GO terms with the highest gene ratio (top 100) is displayed for clearer representation. The terms are ordered by significance, representing their adjusted p-value (x-axis), and the expressed genes were used as the background in this analysis. G. Stacked bar plot of the inferred cellular composition of neural progenitors for each condition. The cell deconvolutional analysis was carried out using CIBERSORTx, and the gene signatures of the neural progenitors were retrieved from the data generated in Belenguer et al., 2021.

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Transcriptomic analysis of the RNA-seq data obtained from P2Y 13 overexpressing cells in vivo . A. Scheme of the experimental design: right panel created with BioRender.com. B. A principal component analysis (PCA) of the most variably expressed (top 3,000) genes of the samples profiled, colored by condition: control ( FO_C , blue), P2Y 13 overexpression ( FO_Y , green). C. Heat-map of the top 1,000 most variably expressed genes across the samples profiled. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. D. Heat-map of the genes associated with the activation/quiescence equilibrium and self-renewal. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. E. Volcano plot showing the differential expression of genes between P2Y 13 overexpressing and control samples (n=3, PY 13 and 3 controls). Differentially expressed genes (DEGs, adjusted p-value <0.05) are in red and green when upregulated or downregulated in the P2Y 13 overexpressing samples, respectively. The bar plot represents the number of DEGs. F. Overrepresentation of gene ontology (GO) terms for biological processes from the DEGs (adjusted p-value <0.05) between the P2Y 13 overexpressing and control samples. A customized selection (18) of significant GO terms with the highest gene ratio (top 100) is displayed for clearer representation. The terms are ordered by significance, representing their adjusted p-value (x-axis), and the expressed genes were used as the background in this analysis. G. Stacked bar plot of the inferred cellular composition of neural progenitors for each condition. The cell deconvolutional analysis was carried out using CIBERSORTx, and the gene signatures of the neural progenitors were retrieved from the data generated in Belenguer et al., 2021.

Techniques: RNA Sequencing Assay, In Vivo, Control, Over Expression, Expressing, Activation Assay, Selection, Generated

Neurogenic trees from the ventral wall of the SEZ tracked in culture following treatment with the P2Y 13 agonist 2MesADP, in the presence or absence of the MRS2211 antagonist. A Representative symmetric neurogenic tree obtained in control conditions after 6 days in culture (N, neuron; X, cell death). B Complex symmetric neurogenic trees of 5 rounds of division obtained in the presence of 2MeSADP after 6 days in culture (N, neuron; X, cell death). The phase contrast images in both A and B depict the lineage progression in the live imaging experiment (day-hour-min). The last images show post-imaging immunocytochemistry of the neuroblast progeny (βIII-tubulin in green) Scale bar 30 µm. C Clones undergoing 1-6 rounds of division in the live imaging experiments (n=5) (n=4 for MRS2211). D Summary of all the clones tracked undergoing 4 or 5 rounds of division in our live imaging experiments, either in control conditions, or when exposed to 2MeSADP or 2MeSADP + MRS2211 (n=5) (n=4 for MRS2211). E Cell survival in the lineage trees (n=5). In all cases the progeny generated were identified by post-imaging immunocytochemistry. All graphs show the mean ±SEM: *p<0.05, and **p<0.01 (ANOVA with a Tukey’s post-test).

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Neurogenic trees from the ventral wall of the SEZ tracked in culture following treatment with the P2Y 13 agonist 2MesADP, in the presence or absence of the MRS2211 antagonist. A Representative symmetric neurogenic tree obtained in control conditions after 6 days in culture (N, neuron; X, cell death). B Complex symmetric neurogenic trees of 5 rounds of division obtained in the presence of 2MeSADP after 6 days in culture (N, neuron; X, cell death). The phase contrast images in both A and B depict the lineage progression in the live imaging experiment (day-hour-min). The last images show post-imaging immunocytochemistry of the neuroblast progeny (βIII-tubulin in green) Scale bar 30 µm. C Clones undergoing 1-6 rounds of division in the live imaging experiments (n=5) (n=4 for MRS2211). D Summary of all the clones tracked undergoing 4 or 5 rounds of division in our live imaging experiments, either in control conditions, or when exposed to 2MeSADP or 2MeSADP + MRS2211 (n=5) (n=4 for MRS2211). E Cell survival in the lineage trees (n=5). In all cases the progeny generated were identified by post-imaging immunocytochemistry. All graphs show the mean ±SEM: *p<0.05, and **p<0.01 (ANOVA with a Tukey’s post-test).

Techniques: Control, Imaging, Immunocytochemistry, Clone Assay, Generated

$Effect of the P2Y 13 receptor on NSC self-renewal. A . Quantification of the symmetric lineage trees (generating only neuroblasts) and the asymmetric lineage trees (generating neuroblasts and new NSCs through self-renewal events) in control conditions, or on exposure to 2MeSADP or 2MeSADP + MRS2211 (n=4). B. Proportion of GFAP + /SOX2 + NSCs relative to the total number of cells in the culture according to the following labels: “Initial” indicates the proportion of GFAP + /SOX2 + in the culture and the “Quiescent” cells are the fraction of the initial cells that remain quiescent throughout the live imaging experiments. The “Clones” reflect the fraction of the initial cells that undergo lineage progression (Control and 2MeSADP n=5 and 2MeSADP+MRS2211 n = 4). C. The models represent the stereotypic behavior of NSCs under control conditions and when exposed to 2MeSADP or 2MeSADP+MRS211. Note how 2MeSADP increases the speed in the cell cycle while impeding self-renewal divisions. By contrast, MRS2211 instructs NSCs to remain quiescent. D. Cell Cycle length within the complex trees tracked (≥4 rounds of division). E . Cell Cycle length for the first division within the complex trees tracked (≥4 rounds of division, n= 5). Note that this first division is normally associated with slow-dividing astroglia, yet 2MeSADP significantly increase the speed of the cell cycle (n=5). All the graphs show the mean ±SEM: *p<0.05, **p<0.01 (T-test for D, E and ANOVA with a Tukeys post-test for B ).$

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Effect of the P2Y 13 receptor on NSC self-renewal. A . Quantification of the symmetric lineage trees (generating only neuroblasts) and the asymmetric lineage trees (generating neuroblasts and new NSCs through self-renewal events) in control conditions, or on exposure to 2MeSADP or 2MeSADP + MRS2211 (n=4). B. Proportion of GFAP + /SOX2 + NSCs relative to the total number of cells in the culture according to the following labels: “Initial” indicates the proportion of GFAP + /SOX2 + in the culture and the “Quiescent” cells are the fraction of the initial cells that remain quiescent throughout the live imaging experiments. The “Clones” reflect the fraction of the initial cells that undergo lineage progression (Control and 2MeSADP n=5 and 2MeSADP+MRS2211 n = 4). C. The models represent the stereotypic behavior of NSCs under control conditions and when exposed to 2MeSADP or 2MeSADP+MRS211. Note how 2MeSADP increases the speed in the cell cycle while impeding self-renewal divisions. By contrast, MRS2211 instructs NSCs to remain quiescent. D. Cell Cycle length within the complex trees tracked (≥4 rounds of division). E . Cell Cycle length for the first division within the complex trees tracked (≥4 rounds of division, n= 5). Note that this first division is normally associated with slow-dividing astroglia, yet 2MeSADP significantly increase the speed of the cell cycle (n=5). All the graphs show the mean ±SEM: *p<0.05, **p<0.01 (T-test for D, E and ANOVA with a Tukeys post-test for B ).

Techniques: Control, Imaging, Clone Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Expressing, Quantitative RT-PCR, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Immunocytochemistry, Incubation

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: CRISPR, Staining

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Over Expression, Injection, Positive Control, Expressing

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Over Expression, Injection

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: RNA Sequencing Assay, In Vivo, Control, Over Expression, Expressing, Activation Assay, Selection, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Control, Imaging, Immunocytochemistry, Clone Assay, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Techniques: Control, Imaging, Clone Assay

Review

anti rabbit p2y 6 r polyclonal antibody (Alomone Labs)

Structured Review

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1) Product Images from "Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis"

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