Journal: Frontiers in Neuroscience

Article Title: Ankk1 Loss of Function Disrupts Dopaminergic Pathways in Zebrafish

doi: 10.3389/fnins.2022.794653

Figure Lengend Snippet: Drd2 immunohistochemistry in adult zebrafish brain. On the right, drd2 protein distribution in transverse sections of zebrafish brain, ankk1 +/+ and ankk1 27 ins/ 27 ins . On the left, schematic depiction of zebrafish brain, transversal section [adapted from ]. (a–e) Boxes on the schematic depictions represent the region of the brain showed by the corresponding immunohistochemistry on the right; (a’–b”) forebrain drd2 staining; (c’–e”) midbrain drd2 staining. Scale bars: (c”,d”,e’) , 50 mm; (a’–e’) , 100 mm; (b”) , 200 mm. Arrows indicate anti-drd2 positive cells.

Article Snippet: Slides were subsequently incubated with anti-ankk1 Rabbit pAb (A16178, ABclonal), or anti-drd2 Rabbit pAb (A12930, ABclonal), 1:200 in BS, ON at 4°C.

Techniques: Immunohistochemistry, Staining

Journal: Stem Cell Reports

Article Title: Dopamine D2 Receptor-Mediated Regulation of Pancreatic β Cell Mass

doi: 10.1016/j.stemcr.2016.05.015

Figure Lengend Snippet: Loss-of-Function and Gain-of-Function Studies of the Dopamine D2 Receptor DRD2 in MIN6 Cells The effects of DPD and dopamine (DA) in control (A, B, C, G) and Drd2 -knockdown (D, E, F) or Drd2 -overexpressing (H, I) MIN6 cells. (A) The number of MIN6 cells per well after 4 days of chemical treatment (days 3–7). Dopamine treatment decreased the number of cells, whereas DPD or cAMP treatment increased the number of MIN6 cells (five independent experiments, each with three to six replicates). (B) DPD increased proliferation of MIN6 cells revealed by EdU incorporation (48 hr) assay (four independent experiments, each with four replicates). (C) Relative cAMP levels after 6 hr of chemical treatment on day 3. DPD increased intracellular cAMP levels, whereas dopamine decreased intracellular cAMP levels, and DPD reversed the dopamine-mediated decrease. (D–F) Drd2 knockdown increased cAMP levels and phenocopied DPD effects. (D) Upper panel: a schematic of the lentiviral vector used for Drd2 shRNA- mRFP expression. Lower panel: fluorescence and transmission images of the Drd2 -knockdown MIN6 cells (D2KD). (E) D2KD cells showed a partial decrease in Drd2 expression (two independent experiments, each with three replicates). (F) Responses of D2KD cells to dopamine chemicals. DPD increased cell number for 9 days culture, but not in D2KD cells. The effect of dopamine decreased in knockdown cells (four independent experiments, each with four replicates). (G–I) Dopamine treatment of wild-type (G) or Drd2 -overexpressing (I) MIN6 cells on days 3–7. Dopamine dose-dependently increased the number of apoptotic cells (G) (three independent experiments, each with four replicates). Upper panel in (H) shows a schematic of the FLAG-tagged Drd2- expressing vector, and lower panel shows RT-PCR analysis of the exogenously overexpressed Drd2 in MIN6 cells. MIN6 cells overexpressing Drd2 (D2-OE) showed increased sensitivity to dopamine. Up to 37% of the cells became apoptotic after treatment with 10 μM dopamine (I) (three independent experiments, each with three replicates). ∗∗ p < 0.01, ∗ p < 0.05 compared with control DMSO-treated cells. Data represent mean ± SD. Student's t test. Scale bar, 100 μm.

Article Snippet: Primary antibodies used were: rabbit anti-DRD2 receptor antibody (1:500; ABBIOTEC, #251384) or mouse monoclonal (7FG-G5-A2) to ADORA2A (1:500; Abcam, #ab79714).

Techniques: Control, Knockdown, Plasmid Preparation, shRNA, Expressing, Fluorescence, Transmission Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Reports

Article Title: Dopamine D2 Receptor-Mediated Regulation of Pancreatic β Cell Mass

doi: 10.1016/j.stemcr.2016.05.015

Figure Lengend Snippet: Synergistic Effects of NECA, an ADORA2A Agonist, and DPD on β Cell Proliferation and Cell Death through Interaction between DRD2 and ADORA2A (A and B) DRD2 and ADORA2A are expressed and form heterodimer in dissociated mouse pancreatic β cells. (A–A″) Islets were dissociated and Duolink assay was performed immediately after dissociation. (B) Co-immunoprecipitates of the dissociated islet cells using anti-DRD2 (IP-D2), anti-ADORA2A (IP-A2A), or mouse IgG (mIgG) antibody were immunoblotted with anti-ADORA2A (IB-A2A) or anti-DRD2 Ab (IB-D2), respectively. The results indicated that DRD2 and ADORA2A formed a heterodimer. (C–G) DRD2-ADORA2A dimerization states detected by Duolink assay were changed by dopamine (DA), DPD, or NECA, an ADORA2A agonist (C–F′). Dissociated islet cell cultures on day 3 were treated with chemicals for 6 hr and subjected to Duolink assay. (G) DRD2-ADORA2A heterodimer formation was suppressed by DPD or NECA, and enhanced by dopamine. (H–J) The effect of NECA on β cell proliferation was examined using islet dissociation cultures. (H) The number of Ins + cells after 4 days of chemical treatment (days 3–7) were counted and was increased by NECA treatment. The dopamine-mediated reduction in the number of β cells was reversed by treatment with NECA or DPD. (I) The number of EdU + among the Ins + cells after 4 days of chemical treatment (days 3–7) were counted. EdU incorporation (48 hr) in β cells was increased by NECA treatment. Decreased β cell proliferation was reversed by treatment with NECA or DPD. (J) β cell apoptosis was assessed after 2 days of chemical treatment (days 3–5). The number of apoptotic β cells was decreased by treatment with NECA or DPD. Dopamine-triggered apoptosis was rescued by DPD but not by NECA. (K) Schematic of crosstalk between dopamine-DRD2 and adenosine-ADORA2A through heterodimer formation. Data represent mean ± SD. All data are obtained from three independent experiments, each with three replicates. ∗∗ p < 0.01, ∗ p < 0.05 compared with control (open bars), Student's t test.

Article Snippet: Primary antibodies used were: rabbit anti-DRD2 receptor antibody (1:500; ABBIOTEC, #251384) or mouse monoclonal (7FG-G5-A2) to ADORA2A (1:500; Abcam, #ab79714).

Techniques: Control