Journal: International Journal of Molecular Sciences

Article Title: P2Y 1 and P2Y 12 Receptors Mediate Aggregation of Dog and Cat Platelets: A Comparison to Human Platelets

doi: 10.3390/ijms26031206

Figure Lengend Snippet: Presence of P2Y 1 and P2Y 12 in dog, cat, and human platelets. ( A , B ) PRP was isolated from the whole blood of dogs, cats, or humans to obtain platelets. Whole lysates of platelets were analysed by immunoblotting using an ( A ) anti-P2Y 1 or ( B ) anti-P2Y 12 receptor antibody (top panels) or ( A , B ) anti-actin antibody (bottom panels). Arrows indicate ( A ) P2Y 1 or ( B ) P2Y 12 receptors. Results are representative of three independent experiments.

Article Snippet: Rabbit anti-human anti-P2Y 1 (cat. No. APR-021) and rabbit anti-human anti-P2Y 12 (cat. No. APR-020) polyclonal antibodies were from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Western Blot

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

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.

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, 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

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.

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: 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

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).

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: 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

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).

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: 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

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).

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: 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

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.

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: 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

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).

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: 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

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 ).

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: Control, Imaging, Clone Assay

Journal: bioRxiv

Article Title: Platelet-specific P2Y 1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide

doi: 10.1101/2024.11.04.621858

Figure Lengend Snippet: Tissue taken from pups of PRYC x PRYP parents was used in PCR to determine offspring homozygous for P2Y 1 - LoxP flanked allele and hemizygous for PF4- cre (test mice) and offspring homozygous for P2Y 1 - LoxP flanked allele and a non carrier for PF4- cre (control ‘wild type’ mice). Representative PCR for LoxP and cre is shown of a litter ( A ). Platelets were taken via cardiac puncture and stained for with anti-CD41-PE conjugated antibody (platelet marker) and anti-P2Y 1 -FITC conjugated antibody to elucidate P2Y 1 expression between ‘test’ (Plt-P2Y 1 -/- ) and control (WT) platelets ( B ). In other experiments, platelets were harvested from blood, and leukocytes harvested from bone marrow of donor mice and their ability to migrate to fMLP (30nM) was measured using a transwell system ( C ) platelets, co-incubated with 100nM ADP, ( D ) neutrophils. Data expressed as means +/-SEM. n=3 ( B ) or 5-6 ( C , D ) per group. Significant difference represented: * P <0.05, ** P <0.01.

Article Snippet: For each sample, 50 μL of PRP was stained with 1 μL of PE-anti-CD41 antibody (BD Pharmingen, 558040, 0.2 mg/mL) and 1 μL of FITC-anti-P2Y 1 antibody (Alomone Labs, APR-021-F, 0.2 mg/mL) antibodies for 30 min at 4°C in the dark.

Techniques: Control, Staining, Marker, Expressing, Incubation

Journal: Purinergic Signalling

Article Title: Machine learning-aided search for ligands of P2Y 6 and other P2Y receptors

doi: 10.1007/s11302-024-10003-4

Figure Lengend Snippet: Comparison of functional effects (in a FLIPR assay of calcium transients) at the hP2Y 1 R stably expressed in 1321N1 astrocytoma cells. A, B Effects of ABBV-744 and several other ML-selected compounds on concentration-dependent hP2Y 1 R activation by selective nucleotide agonist MRS2365: A 30 µM ABBV-744, AZD3695, and AZD5423; B 100 µM ABBV-744, AZD3695, and AZD5423. C Effects of ABBV-744 and ABBV-075 on the activation of the hP2Y 1 R

Article Snippet: Selective P2Y 1 R agonist MRS2365 ([[(1 R ,2 R ,3 S ,4 R ,5 S )-4-[6-amino-2-(methylthio)-9 H -purin-9-yl]-2,3-dihydroxybicyclo-[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt) was from Tocris (Minneapolis, MN).

Techniques: Comparison, Functional Assay, Stable Transfection, Concentration Assay, Activation Assay