Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Expression of P2Y 1 and P2Y 2 receptors in human anagen hair follicles. a P2Y 1 receptors were found in the outer root sheath ( ORS ) and bulb of anagen hair follicles in longitudinal section but not in the inner root sheath ( IRS ). Scale bar = 40 μm. b In transverse section, P2Y 1 receptors were only seen in the outer root sheath ( ORS ). Scale bar = 40 μm. c P2Y 2 receptors were found in the cortex ( CTX ). Scale bar = 40 μm. d Transverse section of anagen hair follicle: P2Y 2 receptors were seen in the cortex ( CTX ) but not in the central medulla ( M ), inner ( IRS ) or outer root sheaths ( ORS ), or in the surrounding adventitial layer ( A ). Scale bar = 40 μm. e , f Controls: the immunoreaction was abolished after preabsorption of the e P2Y 1 and f P2Y 2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars = 100 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Double labelling of P2Y 1 and P2Y 2 receptors with markers for cellular proliferation, and double labelling of P2X 5 receptors with markers for keratinocyte differentiation in anagen hair follicles. a Double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker for proliferating cells ( green ), to show that P2Y 1 receptors are found in proliferating basal cells in the outer root sheath ( ORS ) and bulb region of the hair follicle in longitudinal section. Scale bar = 50 μm. b Transverse section: double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker ( green ), to show that P2Y 1 receptors are found in proliferating cells in the outer root sheath ( ORS ) of the hair follicle. Scale bar = 50 μm. c Longitudinal section of anagen hair follicle through the dermal papilla ( DP ): double labelling of P2Y 2 receptors ( red ) with proliferating cell nuclear antigen (PCNA), a marker for proliferating cells ( green ), showed that P2Y 2 receptors were found in the cortex ( CTX ) and at the edge of the medulla ( M ) but not in the central medulla. P2Y 2 receptors were not found in the matrix ( Ma ), where cells were positive for PCNA. Scale bar = 75 μm. d Longitudinal section of anagen hair follicle: P2Y 2 receptors were absent from the keratinised cuticle ( Cu ) of the hair shaft. PCNA was also found in cells of the outer root sheath ( ORS ). Scale bar = 75 μm. e Double labelling of P2X 5 receptors ( red-brown ) with involucrin, a marker for differentiating cells ( green ). Involucrin was expressed both in the inner root sheath ( IRS ), cortex ( CTX ) and in the outermost edge of the medulla ( M ). P2X 5 receptors were expressed in the inner ( IRS ) and outer root sheaths ( ORS ) and in the medulla ( M ) and matrix cells ( Ma ). There was yellow colocalisation with P2X 5 receptors in the inner root sheath and in cells at the outermost edge of the medulla ( arrow ). The cortex only stained positive for involucrin, not P2X 5 receptors. Scale bar = 50 μm. f Transverse section: double labelling of P2X 5 receptors ( red ) with involucrin ( green ). Involucrin was expressed in the inner root sheath ( IRS ) and cortex ( CTX ) and colocalised ( yellow ) with P2X 5 receptor staining in the inner root sheath ( IRS ). Scale bar = 50 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Marker, Staining

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

Techniques: Control, Staining, Marker, Expressing, Incubation