Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Bar graphs comparing the relative distributions of A 1 R(A1)- and P2Y 2 R(Y2)-immunoreactive elements in each brain region (A-C) and in transfected HEK293T cells (D) . The P2Y 2 R-P2Y 2 R, A 1 R-A 1 R and A 1 R-P2Y 2 R dimers are indicated by Y2-Y2, A1-A1 and A1-Y2, respectively. Total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Raw data are shown in the tables under the graphs. Data are means of three independent experiments.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-localization of A 1 R and P2Y 2 R . A-C . Confocal images of double immunostained Myc-P2Y 2 R (A; green), HA-A 1 R (B; red), and their merge (C; yellow) in co-transfected HEK293T cells. The co-localization of HA-A 1 R and Myc-P2Y 2 R is evident at the cell surface membrane (small arrow). D-L . Confocal images of double immunofluorescence staining in several rat brain regions. P2Y 2 R (D, G, J; red) and A 1 R (E, H, K; green) immunoreactivities were detected in Purkinje cells (D-F), cerebellar nuclei (G-I), and hippocampal CA3 pyramidal cells (J-L). Co-localizations of A 1 R and P2Y 2 R (F, I, L; yellow) were detected in the soma (large arrows) of all tissues, in dendrites of the Purkinje cells, and in neurons of the cerebellar nuclei (arrowheads). Yellow bar indicates 500 μm (A-C) and white bar indicates 100 μm (D-L). Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) each 10-μm optical slice consisted of a stack of 20 0.5-μm thick sections. Serial optical sections were recorded using an air objective lens of (40×, numerical aperture; 0.6).

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection, Double Immunofluorescence Staining, Confocal Laser Scanning Microscopy

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-immunoprecipitation of A 1 R from cerebral cortex (lane 1), cerebellum (lane 2), and hippocampus (lane 3) . Immunoblotting analyses of extracts of rat brain with anti-A 1 R (A), anti-P2Y 2 R (B), and anti-P2Y 2 R with the control peptide of P2Y 2 R (C). Membrane extracts from each region were immunoprecipitated with anti-A 1 R, and analyzed by immunoblotting with anti-P2Y 2 R (D), anti-P2Y 2 R with the control peptide of P2Y 2 R (E), anti-A 1 R (F). No bands were seen in C, E demonstrating the specificity of the antibodies. It was confirmed that immunoprecpitation without primary antibody resulted in no detectable receptor bands in the immunoblotting (data not shown). Approximate molecular masses are shown in kDa.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Immunoprecipitation, Western Blot

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Immunogold electron microscopy of A 1 R and P2Y 2 R visualized using nanogold particles in transfected HEK293T cells (A-D) and rat brain (E-G) . A: Localization of HA-A 1 R (large particles) detected with anti-HA in HA-A 1 R-transfected HEK293T cells. B: Localization of Myc-P2Y 2 R (small particles) detected with anti-Myc in Myc-P2Y 2 R-transfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of HA-A 1 R and Myc-P2Y 2 R in co-transfected HEK293T cells. D: HA-A 1 R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. E-G: Localization of A 1 R and P2Y 2 R in cortical pyramidal cells (E), Purkinje cells (F), and hippocampal pyramidal cells (G) detected with both anti-A 1 R and anti-P2Y 2 R. Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Electron Microscopy, Transfection, Incubation

Journal: Molecular Vision

Article Title: Changes in melatonin receptor expression in a murine model of glaucoma

doi:

Figure Lengend Snippet: Temporal pattern of MT 2 mRNA expression and cellular distribution of MT 2 receptor in ciliary processes of C57BL/6J versus DBA/2J mice. A : Total RNA from the ciliary processes of either control (C57BL/6J) or glaucomatous (DBA/2J) animals at 3, 6, 9, or 12 months of age was extracted, and MT2 mRNA was quantified with quantitative real-time PCR (qPCR) as described in the Methods section. Values were normalized to the content of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Results are the mean ± standard error of the mean (SEM) of 24 animals of each strain (***p<0.001 versus the same mouse strain; # p<0.05, ### p<0.001 versus a different mouse strain; one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test). B : Immunofluorescence images of ciliary processes from 3-month-old C57BL/6J (I), 3-month-old DBA/2J (II), and 12-month-old DBA/2J (III) mice labeled with antibodies against the MT 2 receptor (green). Nuclei were counterstained with propidium iodide (red). Phase-contrast and confocal images show that the MT 2 receptor is mainly located in the non-pigmented epithelium of the ciliary processes, and expression of the receptor is strongly increased in the 3-month-old DBA/2J mice compared to the older mice and to the 3-month-old C57BL/6J mice. Scale bar = 20 μm.

Article Snippet: Then, the following primary antibodies diluted in PBS containing 0.1% TX-100 were incubated at 4 °C overnight: goat anti-MT 1 (Santa Cruz Biotechnologies, Inc. Santa Cruz, CA; sc-15204, 1:100), goat anti-MT 2 (Alomone Labs, Jerusalem, Israel; APR-010, 1:100), and goat anti-GPR50 (Santa Cruz Biotechnologies; sc-15215, 1:75).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions

doi: 10.1101/2022.10.24.513477

Figure Lengend Snippet: A RNAscope in-situ hybridisation of P2Y 2 mRNA expression (magenta) in tumour and matching normal adjacent tissue. B P2Y 2 mRNA expression in tumour (TCGA) and normal (GTEx) pancreatic tissue samples (* p <0.0001). Graph generated using GEPIA. C Kaplan-Meier plot comparing patients with high vs low expression of P2Y 2 in the PAAD TCGA cohort. Graph generated using KMplot. D Top 4 results of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the ‘Molecular Function’ Gene Ontology (GO) functional database. E Incucyte images of the pancreatic cancer cell line AsPC-1 12 hours after treatment with 100 μM ATP alone or with 5 μM AR-C (P2Y2 antagonist). Cells are transduced with Lifeact to visualise f-actin (green). F Schematic of the amino acid sequence of P2Y 2 showing an RGD motif in the first extracellular loop (image generated in gpcrdb.org ). G IF staining of P2Y 2 (green), integrin αV (red) and DAPI (blue) in AsPC-1 cells showing colocalization of P2Y 2 and integrin αV (yellow).

Article Snippet: DNA labelling of anti-aV antibody (P2W7, Santa Cruz) and anti-P2Y2 receptor antibody (APR-010, Alomone labs) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( Simoncelli et al ., 2020 ; Joseph et al ., 2021 ).

Techniques: In Situ, Hybridization, Expressing, Generated, Functional Assay, Transduction, Sequencing, Staining

Journal: bioRxiv

Article Title: Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions

doi: 10.1101/2022.10.24.513477

Figure Lengend Snippet: A Schematic diagram of the hanging drop sphere model for 3D sphere invasion assays. B Brightfield and fluorescent images of spheres formed using AsPC-1 cells (magenta) with a histone 2B (H2B) tagged with red fluorescent protein (RFP) and the stellate cell line PS-1 (green) with H2B tagged with a green fluorescent protein (GFP). Middle pannel shows AsPC-1 cells in spheres with a dotted line highlighting the central sphere area. Spheres were treated with vehicle control or 100 μM ATP alone or with 5 μM AR-C or 10 μM cRGDfV. The quantification is shown in C using SuperPlots, where each colour represents a repeat and the larger points represent the mean % Invasion for each repeat. D Quantification of spheres formed by AsPC-1 cells transfected with a control siRNA or P2Y 2 siRNA and treated with or without 100 μM ATP. E Brightfield and fluorescent images of spheres formed by AsPC-1 cells subjected to CRISPR/Cas9 gene disruption using a control guide RNA (CTR CRISPR ) or P2Y 2 guide RNAs (P2Y 2 CRISPR ) and treated with or without 100 μM ATP. Quantification in F . G, I Brightfield and fluorescent images of AsPC-1 P2Y 2 CRISPR cells or PANC-1 cells (respectively) transfected with wild-type P2RY2 (P2Y 2 RGD ) or mutant P2RY2 D97E (P2Y 2 RGE ) treated with or without 100 μM ATP and its quantification in H and J , respectively. Statistical analysis with Kuskal-Wallis multiple comparison test.

Article Snippet: DNA labelling of anti-aV antibody (P2W7, Santa Cruz) and anti-P2Y2 receptor antibody (APR-010, Alomone labs) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( Simoncelli et al ., 2020 ; Joseph et al ., 2021 ).

Techniques: Transfection, CRISPR, Mutagenesis