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: Biochemical Journal

Article Title: Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

doi: 10.1042/bj20070671

Figure Lengend Snippet: Figure 1 Co-pull-down of the MycHis–P2Y1 and GST–P2Y11 receptors from HEK293 cells

Article Snippet: For Western blot analysis of the P2Y1 receptor knock-down, the nitrocellulose membrane was incubated with an anti-P2Y1 antibody (Alomone) at 1:500 dilution.

Techniques:

Journal: Biochemical Journal

Article Title: Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

doi: 10.1042/bj20070671

Figure Lengend Snippet: Figure 2 Co-immunoprecipitation of the MycHis–P2Y1 and GST–P2Y11 receptor from HEK293 cells

Article Snippet: For Western blot analysis of the P2Y1 receptor knock-down, the nitrocellulose membrane was incubated with an anti-P2Y1 antibody (Alomone) at 1:500 dilution.

Techniques: Immunoprecipitation

Journal: Biochemical Journal

Article Title: Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

doi: 10.1042/bj20070671

Figure Lengend Snippet: Figure 3 Mutation of the MycHis–P2Y11 receptor has no effect on co-pull- down of the MycHis–P2Y1 and GST–P2Y11 receptors

Article Snippet: For Western blot analysis of the P2Y1 receptor knock-down, the nitrocellulose membrane was incubated with an anti-P2Y1 antibody (Alomone) at 1:500 dilution.

Techniques: Mutagenesis

Journal: Biochemical Journal

Article Title: Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

doi: 10.1042/bj20070671

Figure Lengend Snippet: Figure 4 Analysis of the hetero-oligomerization of Cerulean–P2Y1 and Citrine–P2Y11 receptor by FRET experiments

Article Snippet: For Western blot analysis of the P2Y1 receptor knock-down, the nitrocellulose membrane was incubated with an anti-P2Y1 antibody (Alomone) at 1:500 dilution.

Techniques:

Journal: Biochemical Journal

Article Title: Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

doi: 10.1042/bj20070671

Figure Lengend Snippet: Figure 5 Confocal imaging of the internalization of GFP–P2Y1 and GFP– P2Y11 receptors in transfected 1321N1 cells

Article Snippet: For Western blot analysis of the P2Y1 receptor knock-down, the nitrocellulose membrane was incubated with an anti-P2Y1 antibody (Alomone) at 1:500 dilution.

Techniques: Imaging, Transfection

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: 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