Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) mRNA abundance (TPM, transcripts per million) in WT β cells (left), DESeq2-adjusted P values from differential expression analysis in WT versus Bmal1 -/- β cells (middle), and presence or absence of an annotated BMAL1 binding site near genes of putative IVM targets (right). (B) Rhythmic expression of P2ry1 gene in synchronized pseudoislets from WT Beta-TC-6 cells as assessed by quantitative real-time PCR (n=3) (FDR adjusted P value < 0.05).

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Quantitative Proteomics, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) Venn diagram of BMAL1 binding sites identified by ChIP-sequencing overlapping with differentially-expressed genes identified by RNA-sequencing in Bmal1 -/- β -cell line compared to control cell line ( top ). Browser tracks showing decreased expression of P2ry1 gene in Bmal1 -/- cells compared to controls. BMAL1 binding sites upstream of the P2ry1 gene are also indicated ( bottom ). (B) Bioluminescence from WT insulin-NanoLuc pseudoislets in response to 10 µM IVM and/or 10 µM of the P2Y1 antagonist MRS2179 (n=4-8 experiments, 3-8 repeats per experiment). (C) Ratiometric determination of intracellular Ca 2+ using Fura2-AM dye in WT Beta-TC-6 cells stimulated in the presence or absence of 10µM IVM (n=3-8 experiments, 4-12 repeats per experiment). (D) Insulin secretion by ELISA in pseudoislets from P2ry1 KOs and control WT and Bmal1 -/- Beta-TC-6 cells (n=4/genotype/condition). Benjamini and Hochberg FDR-adjusted P values were computed for multiple comparisons following two-way ANOVA. (E) First two principal components (PC1 and PC2) following unbiased principal component analysis (PCA) of DESeq2 normalized counts in WT, WT + IVM, P2yr1 KO, and P2yr1 KO cells (n=4 per group). (F) Mean log 2 -transformed DESeq2-normalized counts in WT, WT + IVM, P2yr1 KO, and P2yr1 KO cells (n=4 per group) at differentially-expressed (1.5 fold, adjusted P value < 0.05) transcripts identified between WT and WT + IVM treated cells). All values represent mean + SEM. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Binding Assay, ChIP-sequencing, RNA Sequencing, Control, Expressing, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) Quantitative real-time PCR screening for disruption of P2ry1 gene expression (n=3-4/genotype) ( top ). Decreased P2Y1 receptor protein expression by Western blot in WT and Bmal1 -/- Beta-TC-6 cells after genetic disruption ( bottom ). (B) Loss of effect of IVM on gene expression in P2ry1 mutant β cells identified by RNA-sequencing (n=4/genotype/condition). Dots represent values that exceed 1.5-fold of the interquartile range. All values represent mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Real-time Polymerase Chain Reaction, Disruption, Gene Expression, Expressing, Western Blot, Mutagenesis, RNA Sequencing

Journal: Brain Sciences

Article Title: Alterations of Purinergic Receptors Levels and Their Involvement in the Glial Cell Morphology in a Pre-Clinical Model of Autism Spectrum Disorders

doi: 10.3390/brainsci13071088

Figure Lengend Snippet: Detailed list of used antibodies and employed protocols for immunohistochemistry.

Article Snippet: P2Y1 , Alomone Labs, Jerusalem, Israel , APR-021-AG , I-ab 1:50 in PBS containing 1% BSA, 0.3% Triton X; incubation for 1 h at RT, and overnight at 4 °C.

Techniques: Immunohistochemistry, Incubation

Journal: Respiratory Research

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

doi: 10.1186/s12931-026-03611-8

Figure Lengend Snippet: Characterization of platelets from C57BL/6J-P2ry1em1Kcl-Tg(Pf4-icre)Q3Rsko (Plt-P2Y1-/- mice. Tissue taken from pups of PRYC x PRYP parents was used in PCR to determine offspring homozygous for P2Y1- LoxP flanked allele and hemizygous for PF4-cre (test mice) and offspring homozygous for P2Y1- 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, or skeletal muscle samples excised and lysed to allow detection of P2Y1R or P2Y12R using western blots using APR-021 antibody ( B ), or APR-009 antibody ( C ). Platelets were also stained with anti-CD41-PE conjugated antibody (platelet marker) and anti-P2Y1-FITC conjugated antibody to elucidate P2Y1R expression between ‘test’ (Plt-P2Y1-/-) and control (WT) platelets ( D ). Data expressed as means +/- SEM. n=3 ( C ). Significant difference represented: *P <0.05, **P <0.01

Article Snippet: FITC- anti-P2Y 1 antibody (Cat #APR-021-F), anti-P2Y 1 antibody (Cat #APR-021), anti-P2Y 1 antibody (Cat #APR-009) and anti-P2Y 12 antibody (Cat #APR-012) was purchased from Alomone Labs. Stromatol (Cat. #321200S) was purchased from Mascia Brunelli, Italy.

Techniques: Tissue, Control, Western Blot, Staining, Marker, Expressing, Negative Control, Polymerase Chain Reaction, FACS

Journal: Journal of neurochemistry

Article Title: ATP inhibits the hypoxia response in type I cells of rat carotid bodies.

doi: 10.1111/j.1471-4159.2004.02978.x

Figure Lengend Snippet: Fig. 4 The inhibitory action of ATP on hypoxia response is mediated via P2Y1 receptors. (a) Dose relation of different purinoreceptor ag- onists on hypoxia response. Plot of the fractional inhibition of the hypoxia-induced [Ca2+]i rise vs. log concentrations of different puri- noreceptor agonists. The potency order for inhibition was 2 MeSATP > ATP > ADP >> a,b-MeATP and UTP, implicating the involvement of P2Y1 receptors. Each cell was subjected first to a hypoxia challenge in standard bath solutions. Following the recovery of [Ca2+]i, a second hypoxia challenge in the presence of purinoreceptor agonist was ap- plied. The protocol was similar to that described in Fig. 2. The amplitude of the hypoxia-induced Ca2+ signal in the presence of the purinoreceptor agonist was compared to that of the first hypoxia challenge. The number of cells included in each data point is shown in parentheses and values of statistical significance (when compared to same concentrations of ATP) are denoted with *. (b) Confocal image of a carotid body section immunostained with a fluorescently labelled antibody against an epitope on the 3rd intracellular loop of the P2Y1 receptors. Note that the fluorescence was localized in the clusters of ovoid shaped type I cells. (c) Confocal image of a carotid body section immunostained with the P2Y1 antibody together with the antigen peptide. In both b and c, the outlines of individual cells were estimated by comparing bright-field and fluorescent images; a spindle-shaped type II cell was marked with an asterisk.

Article Snippet: The sections were then incubated overnight at 4 C with a primary antibody which was a rabbit polyclonal antibody raised against the 3rd intracellular loop of the P2Y1 receptors (1 : 100; Alamone Laboratories, Jerusalem, Israel).

Techniques: Inhibition, Confocal

Journal: Journal of cellular physiology

Article Title: Extracellular beta-nicotinamide adenine dinucleotide (beta-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement.

doi: 10.1002/jcp.22029

Figure Lengend Snippet: Fig. 2. Expression of b-NAD-activated purine receptors P2Y1 and P2Y11 on mRNA and protein levels in HPAEC. (A) The receptor mRNA expressions were examined by Real-Time RT-PCR. Data were calculated relative to internal housekeeping gene (18S rRNA) and are expressed as fold change compared to control W SEM (n U 4). (B) The cell lysates obtained from HPAEC were analyzed by SDS- PAGE followed by immunoblotting using rabbit polyclonal antibodies against P2Y1 and P2Y11. Position of 40 kDa protein marker is shown by arrow. Immunoblotting of b-actin was used as a loading control.

Article Snippet: P2Y1-, P2Y11-, EPAC1-, and Rac1-specific siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Control, SDS Page, Western Blot, Marker

Journal: Journal of cellular physiology

Article Title: Extracellular beta-nicotinamide adenine dinucleotide (beta-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement.

doi: 10.1002/jcp.22029

Figure Lengend Snippet: Fig. 3. Inhibitory analysis (selective antagonists and siRNA-mediated depletion) of the involvement of P2Y1 and P2Y11 receptors in b-NAD-stimulated TER increase. (A) HPAEC were pretreated with either 10 mM MRS2279 (P2Y1 antagonist) or 1 mM NF157 (P2Y11 antagonist) for 30 min prior b-NAD stimulation and used in ECIS assay. Data are representative of several independent experiments (minimum of three) (MP < 0.05 compared with control). (B) RT-PCR analysis of the expression of P2Y1 and P2Y11 mRNAs in the cells treated with scrambled and receptor-specific siRNA. siRNA approach was proved to be very effective in the depletion of P2Y1 and P2Y11 expression. Expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a loading control. (C, D) Depletions of P2Y11 (C) or P2Y1 (D) receptors negatively affect b-NAD-mediated TER response. HPAEC plated in ECIS arrays were transfected with respective scrambled (nsRNA) and siRNA asdescribedinMaterialsandMethods.48 haftertransfection,thecellswereusedinECISassayinthepresenceorabsenceofb-NAD.Time-pointsof b-NAD addition are indicated by arrows. Data are representative of several independent experiments (minimum of three) (MP < 0.05 compared with control).

Article Snippet: P2Y1-, P2Y11-, EPAC1-, and Rac1-specific siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

Journal: Bioengineered

Article Title: Mechanical ventilation induces lung and brain injury through ATP production, P2Y1 receptor activation and dopamine release

doi: 10.1080/21655979.2021.2022269

Figure Lengend Snippet: Mechanical ventilation increased alveolar free ATP and activated P2Y1 receptor. The mice were mechanically ventilated, and the low tidal volume and high tidal volume were set. The mice with spontaneous breathing were used as the control. A: ATP level in mouse BALF detected by luciferase assay, N = 6; B: PY1R expression pattern in lung tissues detected by Western blot, N = 6; C: ATP level in MLE-12 cells detected by luciferase assay; D: PY1R expression pattern in MLE-12 cells detected by Western blot. Cell experiments were conducted 3 times independently. ** p < 0.01. Data in panels A and B were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test, and data in panels C and D were analyzed using t test. ATP, adenosine triphosphate; RLU, relative light unit; BALF, bronchoalveolar lavage fluid; LVT, low tidal volume; HVT, high tidal volume; P2Y1R, P2Y1 receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The primary antibodies included P2Y1R (NBP1-69,246, at a dilution ratio of 1:1000, Novus Biologicals, Littleton, CO, USA), dysbindin-1 (PA5-27,371, at a dilution ratio of 1:1000, Thermo Fisher Scientific), and GAPDH (ab8245, at a dilution ratio of 1:2000, Abcam).

Techniques: Control, Luciferase, Expressing, Western Blot

Journal: Bioengineered

Article Title: Mechanical ventilation induces lung and brain injury through ATP production, P2Y1 receptor activation and dopamine release

doi: 10.1080/21655979.2021.2022269

Figure Lengend Snippet: Activated P2Y1 receptor promoted DA release in mouse hippocampus. The mice were mechanically ventilated, and the low tidal volume and high tidal volume were set. The mice with spontaneous breathing were used as the control. Based on the high tidal volume, mice were performed with 330-min high positive-pressure ventilation. A: DA level in mouse hippocampus detected by ELISA, N = 6; B: dysbindin-1 level in mouse hippocampus detected by Western blot, N = 6; mouse hippocampal neuron cell line HT-22 were treated with P2Y1 receptor activator MRS2365 with cells treated with ACSF as controls, C: DA level in HT-22 cells detected by ELISA; D: levels of P2Y1R and dysbindin-1 in HT-22 cells detected by Western blot. Cell experiments were conducted 3 times independently. ** p < 0.01. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. DA, dopamine; LVT, low tidal volume; HVT, high tidal volume; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACSF, artificial cerebral spinal fluid; P2Y1R, P2Y1 receptor.

Article Snippet: The primary antibodies included P2Y1R (NBP1-69,246, at a dilution ratio of 1:1000, Novus Biologicals, Littleton, CO, USA), dysbindin-1 (PA5-27,371, at a dilution ratio of 1:1000, Thermo Fisher Scientific), and GAPDH (ab8245, at a dilution ratio of 1:2000, Abcam).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Western Blot