Journal: Journal for Immunotherapy of Cancer

Article Title: Pharmacologic targeting of the dopamine D2 receptor impacts the efficacy of immune checkpoint blockade in melanoma

doi: 10.1136/jitc-2025-014080

Figure Lengend Snippet: Graphical abstract. Figure created in BioRender. BRC, bromocriptine; D2R, D2 receptor; HAL, haloperidol; ICI, immune checkpoint inhibitor; IL, interleukin; MCP, metoclopramide; MHC, major histocompatibility complex; PRL, prolactin.

Article Snippet: Samples were loaded in 10% precast polyacrylamide gels (Bio-Rad, cat. #4561036), run at 100 V in Tris/Glycine/sodium dodecyl sulfate (SDS) buffer (Bio-Rad 1610732), transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, cat. #1704272), blocked for 1 hour with 5% dry milk in tris buffered saline-Tween (TBS-T), and probed for PRL (R&D, cat. #AF1445), D2R (Proteintech, cat. #55084-1-AP), and β-Actin-HRP (BioLegend, cat. #664803) in blocking buffer.

Techniques: Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Pharmacologic targeting of the dopamine D2 receptor impacts the efficacy of immune checkpoint blockade in melanoma

doi: 10.1136/jitc-2025-014080

Figure Lengend Snippet: Tumor immune microenvironment in prolactin-locus ICI non-responder and responder models. ( A ) Experimental scheme. B16F0-bearing (CC51xB6)F1 and B6 mice received 100 µg αCTLA-4 and 200 µg αPD-1 on days 3, 6, and 10 after tumor inoculation. On day 13 after tumor inoculation, tumors were enriched for CD45+ TILs then processed for scRNA-seq. ( B ) UMAP and ( C ) composition analysis showing the breakdown of B6U, B6T, CC51U, CC51T annotated cell types overlaying the UMAP ( B ) and proportions of each cell type cluster. For each experimental condition, cell proportions (Y axis) were calculated by dividing the number of cells of a given type by the total number of cells. B6U, B6 untreated; B6T, B6 ICI-treated; CC51U, (CC51xB6)F1 untreated; CC51T, (CC51xB6)F1 ICI-treated. Cell types annotated manually using canonical markers. ( D ) Expression levels of M1 and M2 markers in the MΦ cluster. ( E ) Volcano plot highlighting DEGs in the CD20− CD8+ T cluster between B6T and CC51T. Significant DEG (padj≤0.05, |log2FC | ≥ 1) were determined using FindMarkers and are colored (red=upregulated in B6T; purple=upregulated in CC51T). ( F ) Expression levels of Prlr, Drd2, Htr3a and Htr4 in all clusters. αCTLA-4, anti-cytotoxic T-lymphocyte-associated protein 4; αPD-1, anti-programmed cell death protein-1; B, B cells; B16, B16 F0 cells; CD4+ T, CD4+ T cells; CD20- CD8+ T, CD8+ T cells; CD20+ CD8+ T, CD20+ CD8 T cells; cDC1, conventional dendritic cells, type I; DEGs, differentially expressed genes; Drd2, dopamine D2 receptor; Htr3a, serotonin 5HT3 receptor; Htr4, 5HT4 receptors; ICI, immune checkpoint inhibitor; MΦ, macrophages; MDSC, myeloid-derived suppressor cells; Mono, monocytes; NK T, natural killer T cells; Prlr, prolactin receptor; scRNA-seq, single-cell RNA sequencing; TILs, tumor-infiltrating leukocytes.

Article Snippet: Samples were loaded in 10% precast polyacrylamide gels (Bio-Rad, cat. #4561036), run at 100 V in Tris/Glycine/sodium dodecyl sulfate (SDS) buffer (Bio-Rad 1610732), transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, cat. #1704272), blocked for 1 hour with 5% dry milk in tris buffered saline-Tween (TBS-T), and probed for PRL (R&D, cat. #AF1445), D2R (Proteintech, cat. #55084-1-AP), and β-Actin-HRP (BioLegend, cat. #664803) in blocking buffer.

Techniques: Expressing, Derivative Assay, Single Cell, RNA Sequencing

Journal: Journal of insect physiology

Article Title: Neurochemical regulation of Aedes aegypti salivary gland function.

doi: 10.1016/j.jinsphys.2021.104193

Figure Lengend Snippet: Fig. 5. Immunolocalization of DA receptor in the isolated A. aegypti salivary gland with rat D1- receptor polyclonal antibody. (A) overview of sali vary glands at low magnification with the labels for salivary duct (SD), lateral lobes (LL), proximal lateral lobes (PLL), distal lateral lobes (DLL), median lobe (ML). Square insert in panel A is a representative image (n = 5) of gland treated with secondary anti body only (negative control). Hashtags correspond to the location of the magnified image for the median lobe (B), dorsal lateral lobe (C), proximal lateral lobe (D). Positive staining (green) of the salivary gland with the human D1 antibody was observed in different patterns for all lobes. DAPI counterstaining (blue) was used to localize nuclei. Scale bars are specific for each panel and images are representative of 5 individual replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, the glands were incubated with rat anti-D1 or anti-D2 dopamine receptor polyclonal antibody at a dilution factor of 1:50 (Alamone Labs, Jerusalem, Israel) for 24 h at 4◦.

Techniques: Isolation, Negative Control, Staining

Journal: Journal of insect physiology

Article Title: Neurochemical regulation of Aedes aegypti salivary gland function.

doi: 10.1016/j.jinsphys.2021.104193

Figure Lengend Snippet: Fig. 6. Immunolocalization of DA receptor in the isolated A. aegypti salivary gland with rat D2- receptor polyclonal antibody. (A) overview of sali vary glands at low magnification with the labels for salivary duct (SD), lateral lobes (LL), proximal lateral lobes (PLL), distal lateral lobes (DLL), median lobe (ML). Hashtags correspond to the location of the magnified image for the median lobe (B), dorsal lateral lobe (C), proximal lateral lobe (D). Positive staining (green) of the salivary gland with the human D2 antibody was observed in different patterns for all lobes. DAPI counterstaining (blue) was used to localize nuclei. Scale bars are specific for each panel and images are representative of 5 individual repli cates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, the glands were incubated with rat anti-D1 or anti-D2 dopamine receptor polyclonal antibody at a dilution factor of 1:50 (Alamone Labs, Jerusalem, Israel) for 24 h at 4◦.

Techniques: Isolation, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Role of Dopamine Receptors in the Anticancer Activity of ONC201.

doi: 10.1016/j.neo.2017.10.002

Figure Lengend Snippet: Figure 1. D2-like receptors are expressed and functional in colorectal cancer cells. (A) Assessment of DRD2 mRNA expression in different cancer tissue samples using cBioportal. (B) Viability assessment after 72 hours of treatment with dopamine or sumanirole, as indicated in legend. Western blot analyses of PKA substrate phosphorylation in (C) HCT116 cells treated with 500 μM phosphodiesterase inhibitor IBMX and 10 μM adenylyl cyclase activator forskolin (F), or treated with 10 μM ONC201, in the presence or absence of 1 μM protein kinase A inhibitor peptide 6-22 (PKI); (D) Hep3B cells treated with different doses of ONC201 for 24 hours; (E) HCT116, RKO, and ONC201-resistant RKO cells (RKOr1) treated with 10 μM (HCT116) or 5 μM (RKOp and RKOr1) ONC201 for indicated times.

Article Snippet: Five different lentiviral plasmid shRNA constructs for DRD2 (Origene) were amplified, and DNA was isolated using plasmid mini kit (Qiagen) according to manufacturer's instructions.

Techniques: Functional Assay, Expressing, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Role of Dopamine Receptors in the Anticancer Activity of ONC201.

doi: 10.1016/j.neo.2017.10.002

Figure Lengend Snippet: Figure 3. Transient knockdown of DRD2 in colorectal cancer cells can activate the ISR and reduce cell number. (A) Viable cell count, (B) Western blot analyses of PARP cleavage and CHOP expression, (C) Western blot analyses of ATF4 and CHOP protein expression in cells after DRD2 siRNA-mediated knockdown for 24 hours and subsequent treatment with 10 μM ONC201 for indicated times. *P b .05 versus vehicle-treated scramble siRNA- transfected; P b .05 versus vehicle-treated DRD2 siRNA-transfected. (D) qRT-PCR analyses of DRD1 and DRD2 mRNA expression were performed to verify knockdown and monitor for potential compensatory overexpression of DRD1 receptor. Data are means ± SE from three biological replicates. P b .05 versus viability of control shRNA cells similarly treated. (E) Western blot analyses for PKA substrate phosphorylation in stably transfected control and DRD2 shRNA cells treated with 10 μM ONC201 for 24 hours. (F) Cell proliferation rate assessment of control and DRD2 shRNA-transfected cells was performed by enumerating cell number after indicated times of cell culture. Data are means ± SE from three biological replicates. P b .05 versus viability of control shRNA cells similarly treated.

Article Snippet: Five different lentiviral plasmid shRNA constructs for DRD2 (Origene) were amplified, and DNA was isolated using plasmid mini kit (Qiagen) according to manufacturer's instructions.

Techniques: Knockdown, Cell Counting, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Control, shRNA, Phospho-proteomics, Stable Transfection, Cell Culture

Journal: Neoplasia (New York, N.Y.)

Article Title: Role of Dopamine Receptors in the Anticancer Activity of ONC201.

doi: 10.1016/j.neo.2017.10.002

Figure Lengend Snippet: Figure 4. The anticancer effects of ONC201 are distinct from other selective DRD2 antagonists. (A) CTG assay images, (B) apoptosis measured by sub-G1 analyses, and Western blot analyses of (C) PARP cleavage and (D) ATF4 protein expression in different cancer cell types treated with different selective DRD2 antagonists (O: 10 μM ONC201 for HCT116, SKNSH, and U251, and 5 μM ONC201 for RKO; L: 25 μM L741626; R: 10 μM). (E) Nontumorigenic CCD-CON and IMR90 cells and HCT116 and SKNSH cancer cells treated with indicated doses of L-741,626 for 72 hours. *P b .05 versus vehicle treated; P b .05 versus L-741,626 treated.

Article Snippet: Five different lentiviral plasmid shRNA constructs for DRD2 (Origene) were amplified, and DNA was isolated using plasmid mini kit (Qiagen) according to manufacturer's instructions.

Techniques: CTG Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Modulation of huntingtin S421 phosphorylation in a Huntington’s disease mouse model and its detection in nonhuman primate cerebrospinal fluid

doi: 10.1101/2025.07.18.665500

Figure Lengend Snippet: A. Specificity assessment of Ab2174 for pS421 HTT by WB and the SMC assay in purified, recombinant FLQ23 HTT proteins with/without S421 A or D mutations . i) WB probed with Ab2174 polyclonal antibody or MAB2166 antibody reveals pS421-HTT and total HTT levels, respectively. ii) SMC analysis of pS421-HTT levels (MW1-Ab2174) or total HTT levels (MW1-2B7) performed on serial dilutions of recombinant proteins in i). Representative experiment of n=3 is shown. B. Specificity assessment of Ab2174 for pS421 HTT by WB and SMC assay in HEK293T cells overexpressing FL HTT with/without S421 A or D mutations. i) WB probed with Ab2174 polyclonal antibody or MAB2166 antibody to demonstrate the levels of pS421-HTT and total HTT, respectively. Anti-GAPDH was used as loading control. ii) Normalized pS421 signal (MW1-Ab2174 SMC assay) on total HTT levels (MW1-2B7 SMC assay) performed on samples in i). Means and standard deviations from n=3 biological replica. C. Pharmacological modulation of pS421-HTT levels in HEK293T cells transfected with N571 Q55 HTT protein and FLAG-tagged dopamine D2 receptor, treated with D2R agonist (Bromocriptine) or antagonists (Haloperidol, Raclopride). i) WB probed with MAB2166 antibody, Ab2174 polyclonal antibody or FLAG antibody demonstrating total HTT levels, pS421-HTT and the D2 receptor, respectively. Anti-GAPDH used as loading control. ii) Densitometric analysis of WB in i) and normalized pS421 signal (MW1-Ab2174 SMC assay) on total HTT levels (MW1-2B7 SMC assay) performed on the same samples. Means and standard deviations from n=3 biological replica. Statistical analysis by one-way analysis of variance (*P<0.05; **P < 0.01; ***P < 0.005).

Article Snippet: The pCMV6 expression plasmids containing the cDNA coding for C-term Myc-FLAG-tagged dopamine D2 receptor (D2R; catalog #RC213065) and Protein kinase A catalytic subunit (PKA C α ) (PRKACA; catalog #RC210332) were obtained from ORIGENE (Rockville, Maryland, United States).

Techniques: Purification, Recombinant, Control, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: Inhibitory Effects of Trifluoperazine on Peripheral Proinflammatory Cytokine Expression and Hypothalamic Microglia Activation in Obese Mice Induced by Chronic Feeding With High-Fat-Diet

doi: 10.3389/fncel.2021.752771

Figure Lengend Snippet: List of reagents (antibodies and assay kits) in the study.

Article Snippet: Polyclonal rabbit anti-D2R , Biorbyt, Cat# orb10515, RRID:AB_10747533.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: AKT and GSK3β phosphorylation is modulated by D2R. ( a ) Model of D2R modulation of AKT/GSK3β signaling. Binding of dopamine (DA) to the DA D 2 receptor (D2R) recruits β-arrestin-2, a scaffolding protein, along with the kinase AKT and the phosphatase PP2A to the receptor independently of Gα i/o signaling. PP2A dephosphorylates AKT, inactivating the kinase. Phospho-AKT (P-AKT) is responsible for phosphorylating constitutively active GSK-3β, inactivating it. Thus, D2R-mediated AKT inactivation ultimately increases levels of active, non-phosphorylated GSK-3β. (b) D2R knockdown in mouse renal proximal tubule cells (mRPTCs) via D2R siRNA (72 hr) caused a 130% increase in AKT phosphorylation at the catalytic/stimulatory T308 site, relative to the non-silencing (NS) siRNA control. Acute treatment with D2R antagonist sulpiride (1 µM, 6 hr) doubled AKT phosphorylation, relative to the vehicle control. D2R agonist quinpirole (1 µM, 24 hr) reduced AKT phosphorylation by 30% compared with the vehicle control. (c) D2R knockdown by D2R siRNA in mRPTCs caused a 150% increase in GSK3β phosphorylation (P-GSK3β) at the inhibitory S9 site, while acute sulpiride treatment also increased GSK3β phosphorylation by 50% compared with the respective controls. D2R activation by quinpirole decreased GSK3β phosphorylation by 20%, compared with vehicle. Results are represented as the ratio of P-AKT (T308)/total Akt, and P-GSK3β (S9)/total GSK3β and then normalized as percentage of NS siRNA or vehicle (% of control). All results are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. *P < 0.05, ***P < 0.001; n = 3–4/group; Student’s t-test.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Phospho-proteomics, Binding Assay, Scaffolding, Knockdown, Control, Activation Assay

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: D2R modulates β-catenin phosphorylation and TCF/LEF activity. ( a ) siRNA knockdown of D2R (D2R siRNA) in mouse renal proximal tubule cells (mRPTCs) caused a 20% decrease in phosphorylated β-catenin (P-β-catenin, left panel) compared with the non-silencing (NS) siRNA control (72 hr). Acute sulpiride (D2R antagonist) treatment (1 µM, 6 hr) similarly reduced β-catenin phosphorylation by 25% compared with the vehicle control. Conversely, D2R agonism by quinpirole (1 µM, 24 hr) increased β-catenin phosphorylation, relative to the vehicle control (right panel). Results are represented as the ratio of P-β-catenin/total β-catenin, and then normalized as the percentage of NS siRNA or vehicle (% of control). (b) β-catenin-mediated TCF/LEF luciferase reporter activity was significantly increased by 40% via siRNA-mediated D2R knockdown and decreased by 36% via 1 µM quinpirole treatment to stimulate D2R activity; results are normalized to the respective controls. (c) Acute sulpiride treatment significantly increased TCF/LEF promoter activity by 80%, while direct stimulation by Wnt3a (100 ng/ml, 2 hr) more than doubled (224%) TCF/LEF promoter activity, relative to vehicle control. All data are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. *P < 0.05, **P < 0.01; n = 4–5/group, Student’s t-test for ( a , b ); one-way ANOVA and Holm-Sidak post-hoc test for ( c ).

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Phospho-proteomics, Activity Assay, Knockdown, Control, Luciferase

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: DRD2 SNP cluster rs6276, rs6277 decreases D2R expression, modulates β-catenin phosphorylation and increases cell proliferation in vitro . ( a ) Schema of the relative positions of the SNPs rs6276 and rs6277 in the human DRD2 gene. (b) The joint presence of SNPs rs6276 and 6277 in human renal proximal tubule cells reduced D2R mRNA levels by 50% (left panel) along with a concomitant 50% decrease in D2R protein levels (middle panel). Levels of phospho-β - catenin (P-β - catenin) also decreased by 40%, relative to control cells without these SNPs (right panel). Insets show representative immunoblots. (c) Transfection of the DRD2 SNP-containing cells with a human DRD2 plasmid without the SNPs significantly boosted D2R mRNA levels 30-fold (left panel), as well as D2R protein levels 2.2-fold (middle panel). Increasing wild-type D2R expression in these cells also increased P-β - catenin levels (right panel, inset showing western blot), relative to the empty vector control. (d) Presence of DRD2 SNPs rs6276 and rs6277 led to a 2.7-fold increase in cellular proliferation as indicated by the Ki-67 marker. All data are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. **P < 0.01, ***P < 0.001; n = 3–4/group; Student’s t-test.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Expressing, Phospho-proteomics, In Vitro, Control, Western Blot, Transfection, Plasmid Preparation, Marker

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: D2R expression modulates renal cell proliferation in vivo . ( a ) Kidney-selective D2R knockdown in vivo via siRNA (D2R siRNA) decreased β-catenin phosphorylation (P-β-catenin) in mouse renal cortex by 40% relative to the NS siRNA control. (b) D2R siRNA knockdown also increased the expression of the cell proliferation marker Ki-67. Merged confocal images show Ki-67 (red) and DAPI nuclear staining (blue); magenta shows colocalization. Scale bar = 50 µm. (c) Quantification of the Ki-67-positive cells in the renal cortex revealed significant increases in the number of Ki-67-positive cells/field along with a 3-fold increase in Ki-67 mRNA following D2R siRNA treatment compared with control mice treated with the NS siRNA control; n ≥ 4 for all groups. (d) Rescue of D2R expression via AAV-mediated renal D2R overexpression (D2R AAV) reversed the effects of in vivo D2R siRNA on β-catenin phosphorylation by raising P-β-catenin levels (left panel); inset shows a representative immunoblot. Alongside is the schedule for the respective siRNA and AAV treatments. Ki-67 expression was decreased in the D2R AAV-treated kidneys by 50% in comparison with mice treated with control AAV (CAAV, right panel). (e) Confocal immunofluorescence imaging of the D2R AAV rescue of D2R siRNA knockdown showed a 2-fold decrease in Ki-67-positive cells. Merged confocal images show DAPI (blue) and Ki-67 (red); magenta shows colocalization. Scale bar = 50 µm. (f) Accompanying quantification of Ki-67-positive cells in response to D2R AAV rescue of D2R siRNA knockdown. All data are represented as the mean ± SEM. *P < 0.05, **P < 0.01; n = 3–4/group; Student’s t-test.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Expressing, In Vivo, Knockdown, Phospho-proteomics, Control, Marker, Staining, Over Expression, Western Blot, Comparison, Immunofluorescence, Imaging

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: D2R modulates Wnt3a expression. ( a ) D2R siRNA (72 hr) increased Wnt3a mRNA levels 2.6-fold (left panel) in mouse renal proximal tubule cells. Conversely, the D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a expression by 70% in these cells (right panel). ( b ) Kidney-selective D2R knockdown in vivo via renal subcapsular D2R siRNA infusion similarly increased Wnt3a expression 4-fold in mouse renal cortex (left panel). ( c ) Human renal proximal tubule cells with D2R expression-reducing SNPs rs6276 and rs6277 had increased Wnt3a mRNA levels (2.9-fold, left panel) and protein expression (1.5-fold, right panel) in the setting of diminished D2R expression, relative to cells without these SNPs (wild-type, WT). (d) D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a protein by 44% in the SNP-free wild-type (WT) human renal proximal tubule cells compared with vehicle. Acute treatment with D2R antagonist sulpiride (1 µM, 6 hr) significantly increased Wnt3a protein by 180% in the WT human renal proximal tubule cells compared with vehicle. (e) Wnt3a knockdown via siRNA (Wnt3a siRNA) significantly decreased Wnt3a protein levels (right panel) in human renal proximal tubule cells expressing SNP cluster rs6276 and 6277 and partially attenuated the increased levels of proliferation in these cells, as indicated by the decrease in Ki-67 mRNA (right panel). All data are represented as the mean ± SEM. For Panels (a,c–e): all cell experiments were conducted on n ≥ 3 separate experimental dates and performed in triplicate. For Panel (b): all animal experiments used n = 4–5 animals per group; measurements were performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Expressing, Knockdown, In Vivo

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: Modulation of Wnt3a expression via a conserved TCF/LEF promoter site. ( a ) Phylogenetic tree of WNT3A across multiple species: human, mouse, rat, chicken, and zebrafish. The WNT3A exons were used to construct the phylogenetic tree. Evolutionary distances in the units of base substitutions per site were calculated using the maximum composite likelihood method. The phylogenetic tree was then constructed using the Neighbor-Joining method with the sum of branch lengths = 0.645. (b) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish WNT3A promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the transcriptional start site (TSS). (c) Schematic representation of our luciferase reporter of TCF/LEF-dependent WNT3A transcription (left panel). The reporter construct consists of a selected segment of the human WNT3A promoter region containing a highly conserved TCF/LEF site upstream of the Gaussia luciferase gene. We show two variants of the reporter: one containing the wild-type (WT) TCF/LEF site (Human-WT) and another where the TCF/LEF site was inactivated by mutation (Human-Mut). Upon expression of a WNT3A transcriptional reporter in human renal proximal tubule cells, the Human-Mut reporter demonstrated significantly decreased WNT3A promoter activity compared with the WT control (right panel). The data were normalized to alkaline phosphatase expression. *P < 0.05; Student’s t-test. (d) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish DRD2 promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the TSS. (e) Treatment with either LiCl (25 mM, 24 hr) or recombinant Wnt3a (100 ng/ml, 24 hr) significantly increased D2R mRNA expression. Data are expressed as mean ± SEM of 3 experiments conducted on separate experimental dates and performed in triplicate. *P < 0.05; Student’s t-test ( c ). *P < 0.05, ***P < 0.001; one-way ANOVA, Holm-Sidak post-hoc test ( e ).

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Expressing, Construct, Binding Assay, Luciferase, Mutagenesis, Activity Assay, Control, Recombinant

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: D2R overexpression partially reverses increased Wnt3a expression and cell proliferation in response to ischemia/reperfusion injury. ( a ) AAV-mediated D2R overexpression (D2R AAV) in mouse kidney prior to reperfusion caused a 70% decrease in Wnt3a mRNA levels (left panel) and a 40% decrease in Wnt3a protein levels (middle panel). There was also a 30% decrease in Ki-67 mRNA in the renal cortex relative to the control AAV vector (right panel). Alongside is the schedule for the respective ischemia/reperfusion (I/R) and AAV treatments. ( b ) Representative confocal immunofluorescence images of mouse renal cortex following AAV-mediated D2R overexpression in an I/R model of acute renal injury. There was decreased nuclear Ki-67 (red), nuclear β-catenin (green) and decreased nuclear co-localization of Ki-67 and β-catenin with DAPI (blue). Merged images: blue = DAPI, red = Ki-67, green = β-catenin, overlap = white); arrowheads indicate nuclear overlap among Ki-67, β-catenin, and DAPI signals. Scale bar = 50 µm. All data are represented as the mean ± SEM using 4 animals per group. All measurements were performed in triplicate. *P < 0.05, ***P < 0.001, Student’s t-test.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Over Expression, Expressing, Control, Plasmid Preparation, Immunofluorescence

Journal: Scientific Reports

Article Title: Dopamine D 2 receptor modulates Wnt expression and control of cell proliferation

doi: 10.1038/s41598-019-52528-4

Figure Lengend Snippet: Model of cross-talk between D2R and Wnt/β-catenin pathways, and Wnt3a transcriptional regulation. We propose the following mechanism by which D2R and Wnt3a reciprocally regulate their expression: (a) Wnt3a stimulation of the Frizzled (FZD) receptor/LRP5/6 complex leads to less β-catenin degradation via the destruction complex and proteasomal pathway. Instead, more β-catenin is available for translocation into the nucleus where it increases Wnt3a transcription and expression via a TCF/LEF site within the Wnt3a promoter. This leads to a positive feedback loop that amplifies further Wnt3a signaling. (b) Increased nuclear translocation of β-catenin also upregulates D2R expression through TCF/LEF sites within the D2R promoter. Increased D2R expression leads to more β-catenin phosphorylation, resulting in enhanced β-catenin proteasomal degradation and therefore less β-catenin nuclear translocation. This ultimately inhibits further β-catenin-mediated Wnt3a transcription to maintain cellular homeostasis.

Article Snippet: To construct D2R AAV, a 1.7-kb human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid .

Techniques: Expressing, Translocation Assay, Phospho-proteomics