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: 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 Cell and Developmental Biology

Article Title: Nucleus accumbens shell modulates seizure propagation in a mouse temporal lobe epilepsy model

doi: 10.3389/fcell.2022.1031872

Figure Lengend Snippet: Increased excitability of D1R- and D2R-MSNs in the NAc shell after TLE seizures. (A) Schematic diagram of virus microinjection to enable expression of eGFP reporter on D1R-MSNs or D2R-MSNs in the NAc shell and KA microinjection into BLA to induce TLE seizures of C57BL/6 mouse. (B) Schematic diagram of TLE seizure induction followed by in intro electrophysiological recording in the NAc shell. (C) Representative current-clamp recordings. (D) Summary input–output curves of D1R-MSN of the mice treated KA ( n = 15 cells) show increased AP generations in response to current injections, comparing with the control mice treated with ACSF ( n = 18 cells). *** p < 0.001, F = 70.049, df = 1, two-way ANOVA with Tukey post-hoc test. (E–I) Summary data of resting membrane potential (RMP), AP threshold, rheobase current, and membrane input resistance (R in ) of D1R-MSN of the mice treated with ACSF ( n = 18 cells) or KA ( n = 15 cells). ** p < 0.01, *** p < 0.001, Student’s unpaired t-test. (J) Representative current-clamp recordings and (K) summary input–output curves of D2R-MSN of the mice treated KA ( n = 13 cells) show increased AP generations in response to current injections, compared with the control mice treated with ACSF ( n = 15 cells). *** p < 0.001, F = 20.136, df = 1, two-way ANOVA with Tukey post-hoc test. (L–O) Summary data of resting membrane potential (RMP), AP threshold, rheobase current, and membrane input resistance (R in ) of D2R-MSN of the mice treated with ACSF ( n = 15 cells) or KA ( n = 13 cells). ** p < 0.01, *** p < 0.001, student’s unpaired t-test.

Article Snippet: In the experiments investigating functional interaction between D1R-MSN and D2R-MSN, 15 μM SKF 38393 hydrochloride (MedChemExpress #HY-12520 A) was used to activate D1R, 15 μM quinpirole hydrochloride (sigma # Q102) was used to activate D2R, and 100 μM CGP35348 (MedChemExpress #HY-103530) was used to block GABA B receptors.

Techniques: Virus, Microinjection, Expressing, Control, Membrane

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nucleus accumbens shell modulates seizure propagation in a mouse temporal lobe epilepsy model

doi: 10.3389/fcell.2022.1031872

Figure Lengend Snippet: Increased synaptic transmission of D1R-MSN, but not D2R-MSN, in the NAc shell after TLE seizures. (A) Representative sEPSC of D1R-MSN of the mice treated with ACSF or KA. (B) Cumulative probability of the amplitudes and the averaged amplitude of sEPSCs of D1R-MSN of the mice treated with ACSF ( n = 9 cells) or KA ( n = 14 cells). * p < 0.05, Student’s unpaired t-test (the same test was used below in this figure). (C) Cumulative probability of the inter-event intervals of sEPSCs and the averaged frequency of sEPSCs of D1R-MSN of the mice treated with ACSF ( n = 14 cells) or KA ( n = 9 cells). *** p < 0.001. (D) Representative recordings of sIPSC of D1R-MSN of the mice treated with ACSF or KA. (E) Cumulative probability of the amplitudes and the averaged amplitude of sIPSCs of D1R-MSN of the mice treated with ACSF ( n = 9 cells) or KA ( n = 8 cells). (F) Cumulative probability of the inter-event intervals of sIPSCs and the averaged frequency of sIPSCs of D1R-MSN of the mice treated with ACSF ( n = 9 cells) or KA ( n = 8 cells). (G) Representative recordings of sEPSC of D2R-MSN of the mice treated with ACSF or KA. (H) Cumulative probability of the amplitudes and the averaged amplitude of sEPSCs of D2R-MSN of the mice treated with ACSF ( n = 10 cells) or KA ( n = 8 cells). (I) Cumulative probability of the inter-event intervals of sEPSCs and the averaged frequency of sEPSCs of D2R-MSN of the mice treated with ACSF ( n = 10 cells) or KA ( n = 8 cells). (J) Representative recordings of sIPSC of D2R-MSN of the mice treated with ACSF or KA. (K) Cumulative probability of the amplitudes and the averaged amplitude of sIPSCs of D2R-MSN of the mice treated with ACSF ( n = 10 cells) or KA ( n = 10 cells). (L) Cumulative probability of the inter-event intervals of sIPSCs and the averaged frequency of sIPSCs of D2R-MSN of the mice treated with ACSF ( n = 10 cells) or KA ( n = 10 cells).

Article Snippet: In the experiments investigating functional interaction between D1R-MSN and D2R-MSN, 15 μM SKF 38393 hydrochloride (MedChemExpress #HY-12520 A) was used to activate D1R, 15 μM quinpirole hydrochloride (sigma # Q102) was used to activate D2R, and 100 μM CGP35348 (MedChemExpress #HY-103530) was used to block GABA B receptors.

Techniques: Transmission Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nucleus accumbens shell modulates seizure propagation in a mouse temporal lobe epilepsy model

doi: 10.3389/fcell.2022.1031872

Figure Lengend Snippet: Chemogenetic inhibition of D1R-MSN or D2R-MSN in the NAc shell alleviates TLE seizures. (A) Representative recording shows spontaneous AP firings of D1R-MSN and (B) D2R-MSN transfected with DREADD receptor hM4D (Gi) are suppressed by CNO (5 μM) perfusion. Membrane potential was held under current-clamp at around −50 mV. (C) Representative confocal images show the expression of mCherry and eGFP on D1R-MSN transfected with AAV2/9-DIO-hM4D(Gi)-mCherry and AAV2/9-D1-Cre-eGFP viruses. (D) Representative confocal images show the expression of mCherry and eGFP on D2R-MSN transfected with AAV2/9-DIO-hM4D(Gi)-mCherry and AAV2/9-D2-Cre-eGFP viruses. (E) Schematic diagram of experimental design to examine the effects of chemogenetic inhibition of D1R-MSN or D2R-MSN in the NAc shell on TLE seizures induced by intra-basolateral amygdala (intra-amygdala) microinjection of KA. The mice are allocated into four groups: KA group n = 13 mice; CNO + KA group n = 14 mice; CNO + D1-Gi + KA group n = 10 mice; CNO + D2-Gi + KA group n = 15 mice. (F) Racine’s scaling for maximal seizure stage every 10 min during the whole course of 120 min after KA microinjection. * p < 0.05, *** p < 0.001, F = 18.46, df = 3, two-way ANOVA with repeated-measures. (G) Onset time of the first seizure after KA microinjection. One-way ANOVA. (H) Latency to secondary GSs after KA microinjection. ** p < 0.01, one-way ANOVA. (I) Total number of secondary GSs after KA microinjection. *** p < 0.001, one-way ANOVA. (J) Maximal seizure stage in the whole course of 120 min after KA microinjection. ** p < 0.01, Kruskal–Wallis H test with Bonferroni correction (K) Mortality rate after KA microinjection. p > 0.05, Kruskal–Wallis H test. (L) Representative recording and statistical data (M) show AP firing in response to a step current before or after perfusion of 15 μM D1R agonist SKF 38393. n = 21 cells, * p < 0.05, Student’s unpaired t-test. (N) Representative recording and statistical data (O) show AP firing in response to a step current before or after perfusion of 15 μM D2R agonist quinpirole. n = 18 cells, * p < 0.05, Student’s unpaired t-test. (P) Representative recording and statistical data (Q) show AP firing in response to a step current before or after perfusion of 15 μM D1R agonist SKF 38393 in the presence of GABA A/B blockers (10 μM SR95531/100 μM CGP35348). n = 15 cells, p > 0.05, Student’s unpaired t-test. (R) Representative recording and statistical data (S) show AP firing in response to a step current before or after perfusion of 15 μM D2R agonist quinpirole in the presence of GABA A/B blockers. n = 20 cells, p > 0.05, Student’s unpaired t-test. In the figures G–K, the statistical differences are compared between the KA group and the CNO + KA group, and between the CNO + KA group and the CNO + D1R-Gi + KA group or the CNO + D2R-Gi + KA group. n.s means not statistically significant.

Article Snippet: In the experiments investigating functional interaction between D1R-MSN and D2R-MSN, 15 μM SKF 38393 hydrochloride (MedChemExpress #HY-12520 A) was used to activate D1R, 15 μM quinpirole hydrochloride (sigma # Q102) was used to activate D2R, and 100 μM CGP35348 (MedChemExpress #HY-103530) was used to block GABA B receptors.

Techniques: Inhibition, Transfection, Membrane, Expressing, Microinjection

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

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet: (A–F) Dispersal of DAT nanodomains is promoted by the D2R antagonist haloperidol and blocked by the agonist quinpirole and by the N-type VGCC blocker ω-conotoxin. Shown are example varicosities from dSTORM of DA neurons subjected to 5-min treatment with haloperidol (10 nM), haloperidol (10 nM) plus quinpirole (50 μM), haloperidol (10 nM) plus ω-conotoxin (1 μM), or haloperidol (10 nM) plus ω-agatoxin (1 μM). (A) DAT clusters with a diameter greater than 75 nm are shown in green and clusters smaller than 75 nm in magenta. (B) Dense DAT clusters are shown in red (>80 localizations; radius, 50 nm). (C) Localizations segmented by Voronoi tessellation. (D and E) Normalized fraction of DAT clusters greater than 75 nm (percent) (D) and normalized fraction of DAT localizations in dense clusters (percent) (E). Means ± S.E.; one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (F) PDFs (curves are color coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. Data are from 3 cultures and 153 control, 166 haloperidol, 167 quinpirole, 178 ω-conotoxin, and 176 ω-agatoxin varicosities. (G–R) Single-particle tracking dSTORM (SPT-dSTORM) identifies changes in DAT mobility in response to NMDA and D2R antagonism. (G) Example live dSTORM images of DA neurons labeled with the fluorescent cocaine analog DG3–80. (H) Boxed region in (B). (I and J) Detected tracks (I) and distribution of segment lengths (J) in a varicosity from the control SPT-dSTORM experiment (16-ms frame rate). (K) Example traces of a confined and an unconstrained freely moving particle (DG3–80-labeled DAT). (L) Fraction of unconstrained tracks of the total number of tracks. Data are means of 23 tracks ± S.E. (M) PDF for total mean jump distances (MJD) (i.e., the distance an individual molecule/particle has traveled between each image) for control experiments. (N) PDF when separated into static and freely moving particles. Among the freely moving particles, some moved very fast with MJDs of 200–350 nm. (O) NMDA (20 μM, 5 min) increases the population of DAT molecules with MJDs greater than 200 nm, which is blunted by AP5 (100 μM). (P) Fraction of fast-moving particles (>200 nm). Means ± S.E.; data from 13 control, 4 NMDA, and 5 NMDA plus AP5 experiments. (Q) The D2R antagonists haloperidol (10 nM, 5 min) and UH-232 (1 μM) increase the population of DAT molecules with MJDs greater than 200 nm. (R) fraction of fast-moving particles (>200 nm), Means ± S.E.; data are from 10 control, 6 haloperidol, and 5 UH-232 experiments; one-way ANOVA, *p < 0.05; **p < 0.01. See also , and .

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Control, Single-particle Tracking, Labeling

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet: (A) Example dual-color dSTORM images of the D2R (red, CF568) and DAT (blue, Alexa 647) in a DA neuronal extension (left) with a close-up image of a varicosity (right) corresponding to the boxed region in the left image. (B) Example dual-color dSTORM images of varicosities from DA neurons showing localizations for D2R (red) and DAT (blue). Left: control. Right: neurons exposed to haloperidol (10 nM, 5 min). (C–F) Effect of NMDA and haloperidol on D2R clustering. Top panels: control, NMDA (20 μM), and NMDA (20 μM) plus AP5 (100 μM). Bottom panels: control, haloperidol (10 nM), and haloperidol (10 nM) plus quinpirole (50 μM). (C and D) Normalized fraction of D2R localizations in large clusters (>75 nm in diameter) (percent) (C) and normalized fraction of D2R localizations in dense clusters (>80 localizations; radius, 50 nm) (percent). Means ± S.E., one-way ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05. (E) PDFs (color-coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. (F) PDFs (color-coded as in the bar diagrams) of the association of D2R with DAT clusters determined by Voronoi tessellation-based association . Data are from 3 cultures and 112 control, 157 NMDA, and 75 NMDA plus AP5 varicosities and 179 control, 195 haloperidol, and 150 quinpirole varicosities. See also and .

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Control

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet:

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Virus, Recombinant, Synthesized, Generated, Software

Journal: Pharmacology Research & Perspectives

Article Title: Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium‐dependent, non‐D 2 receptor‐dependent, manner

doi: 10.1002/prp2.689

Figure Lengend Snippet: Dopamine D 2 receptor is present and signals in glioma cell lines. A, Western blots indicate the presence of D 2 R in both mature, glycosylated states (~80 kDa) and immature, lower molecular weight species (~47 kDa) in untreated glioma cell lines (quantified in Figure ). B, qrt‐PCR for DRD2 and GAPDH transcripts were carried out in untreated glioma cell lines using standard TaqMan probes. Standard deviations for GAPDH expression were less than 0.085. C, 100‐1000 nmol/L D 2 R agonist sumanirole for 2 hours reduces forskolin‐stimulated cytoplasmic cAMP levels in U87MG, with a IC50 of 250 nmol/L, compared to previously reported EC50 values of 17‐75 nmol/L for cell based assays in D 2 R expressing CHO cells. ³¹ Data are representative of 2‐3 independent experiments in triplicate. * P < .05; **** P < .001 via two‐way ANOVA with Dunnett's post hoc

Article Snippet: To generate these lines, commercially available DRD2 GIPZ‐shRNA viral particles (Dharmacon, Lafayette, CO, USA) or myc‐DDK tagged DRD2 lentiORF particles (Origene Technologies, Rockville, MD, USA) were used according to the manufacturer's recommendations, using 5 μg of particles (titer > 10 7 TU/mL) per 10 cm dish, followed by puromycin selection.

Techniques: Western Blot, Molecular Weight, Quantitative RT-PCR, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium‐dependent, non‐D 2 receptor‐dependent, manner

doi: 10.1002/prp2.689

Figure Lengend Snippet: D 2 R is not involved in cytotoxic responses to D 2 R antagonists. A, 48 hours MTT concentration response curves were generated from 10 nmol/L to 100 µmol/L for thioridazine in U87MG cells in the presence of 0, 10, 50, or 100 µmol/L ropinirole, a D 2 R agonist. The presence of ropinirole did not significantly alter thioridazine sensitivity. B, Cotreatment with 30 µmol/L sumanirole, a D 2 R agonist, did not significantly reduce the induction of apoptosis in U87MG cells treated with 0.3‐100 µmol/L thioridazine for 24 hours and analyzed via annexin V/&‐AAD flow cytometry. C, Cotreatment with 30 µmol/L PHNO, a D 2 R agonist, did not significantly alter sensitivity of U87MG cells to 0.3‐100 µmol/L pimozide according to 48 hours MTT concentration response curves. D, Knockdown of DRD2 by ~50% (Figure D) in U87MG cells via stable lentiviral transduction reduces cell proliferation slightly, but does not significantly affect response to 10 µmol/L thioridazine as measured by cell counts. E, 48 hours MTT concentration response curves are presented for U87MG‐DRDOE and U87MG‐DRD2KD cells under treatment with 0.3‐100 µmol/L thioridazine. No significant differences in response were noted from parental cells. F, 48 hours concentration response curves for U87MG, U87MG‐DRDOE and U87MG‐DRD2KD cells treated with 0.1‐100 µmol/L remoxipride. No significant sensitivity is noted in any cell line used. Data are representative of two independent experiments carried out in triplicate and is analyzed by two‐way ANOVA with Sidak's post hoc. **** P < .0001 via two‐way ANOVA with Sidak's post hoc vs control for series of matched color.; # P < .0001 for matched treatment pairs via two‐way ANOVA with Sidak's post hoc

Article Snippet: To generate these lines, commercially available DRD2 GIPZ‐shRNA viral particles (Dharmacon, Lafayette, CO, USA) or myc‐DDK tagged DRD2 lentiORF particles (Origene Technologies, Rockville, MD, USA) were used according to the manufacturer's recommendations, using 5 μg of particles (titer > 10 7 TU/mL) per 10 cm dish, followed by puromycin selection.

Techniques: Concentration Assay, Generated, Flow Cytometry, Knockdown, Transduction, Control

Journal: Pharmacology Research & Perspectives

Article Title: Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium‐dependent, non‐D 2 receptor‐dependent, manner

doi: 10.1002/prp2.689

Figure Lengend Snippet: D 2 R antagonists, other than remoxipride, alter calcium signaling in glioma cell lines. A, U87MG cells were treated with 0.1‐100 µmol/L thioridazine for 48 hours in the presence or absence of 3 µmol/L fendiline, a nonspecific calcium channel blocker and calmodulin inhibitor. MTT concentration response curves suggest a significant sensitization to thioridazine in cells co‐treated with fendiline. Values shown are normalized to vehicle treated cells and represent three independent experiments. B, 10 µmol/L thioridazine induces a strong and sustained increase in cytosolic calcium release as measured by single cell imaging of U87MG cells preloaded with 2 µmol/L Fura‐2 AM for 30 minutes. This increase is due to both ER calcium release (as shown by the increase in the absence of extracellular calcium, in red) and calcium influx from the extracellular space (2 mmol/L CaCl 2 , in blue). Unlike the positive control, 2 µmol/L thapsigargin (Figure A), the cell is unable to clear this signal for a significant amount of time. Traces are presented as the ratio of fluorescence at 340 and 380 nm. C, Thioridazine increases cytosolic calcium signal in a concentration dependent manner in the presence of 0 or 2 mmol/L CaCl 2 as measured by Fura‐2 AM fluorescence (shown as maximum ratio of fluorescence at 340 and 380 nm) of U87MG cells preloaded with Fura‐2 AM for 30 minutes as measured by FLIPR assay. D, Maximal calcium responses of selected compounds as measured by Fura‐2 AM FLIPR assay are presented. All compounds induced increases in cytosolic [Ca ++ ] in the presence of 2 mmol/L CaCl 2 except for remoxipride. E, Remoxipride does not affect cytoplasmic calcium levels as measured by single cell imaging with Fura‐2 AM as in (B). F, Maximal calcium responses are not significantly altered by overexpression or knockdown of DRD2 in FLIPR imaging of Fura‐2 AM as in C. A is representative of three experiments in triplicate, while single cell imaging experiments are representative of three independent experiments with at least two cover slips and 40 cells. FLIPR assays are representative of at least two independent experiments carried out in quadruplicate. * P < .05; ** P < .01; *** P < .005; **** P < .0001 via two‐way ANOVA with Sidak's post hoc

Article Snippet: To generate these lines, commercially available DRD2 GIPZ‐shRNA viral particles (Dharmacon, Lafayette, CO, USA) or myc‐DDK tagged DRD2 lentiORF particles (Origene Technologies, Rockville, MD, USA) were used according to the manufacturer's recommendations, using 5 μg of particles (titer > 10 7 TU/mL) per 10 cm dish, followed by puromycin selection.

Techniques: Concentration Assay, Imaging, Positive Control, Fluorescence, Over Expression, Knockdown