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 Clinical Medicine

Article Title: Immunohistochemical Comparison of Dopamine-2 Receptor Expression in Resistant and Non-Resistant Prolactinomas

doi: 10.3390/jcm14207344

Figure Lengend Snippet: D2R and Erα receptor IHC of ( a ) D2R staining neurons positive in normal spinal cord (200X); ( b ) 28-year-old male (non-resistant) with tumor cells positive for D2R (400X); ( c ) 34-year-old male (resistant) with tumor cells negative for D2R (400X); ( d ) 31-year-old female (non-resistant) with tumor cells positive for ER (400X); and ( e , f ) 11-year-old pre-pubertal girl with a resistant tumor whose tumor cells were positive for D2R and negative for Erα (( e ) 400X, ( f ) 400X)). Scale bar, 50 μm.

Article Snippet: D2R , Novus Bio , NLS1403 , Rabbit; polyclonal , 1:1000 , AB_523304.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: A2A-D2 Heteromers on Striatal Astrocytes: Biochemical and Biophysical Evidence

doi: 10.3390/ijms20102457

Figure Lengend Snippet: A2A-D2 heterodimers on rat striatal astrocytes: proximity ligation assay. Detection of in situ PLA A2A-D2 heteroreceptor complexes was carried out with primary antibodies (rabbit polyclonal anti-A2AR, mouse monoclonal anti-D2R and goat polyclonal anti-GFAP) in rat striatal slices. ( A ) The merge of the maximum intensity projections of a representative field (240x240 µm; z 10 µm) is shown; GFAP (red), DAPI (blue), PLA for A2A-D2 heteroreceptor complexes appears as yellow clusters. The boxed region (i) is shown at a higher magnification in ( B ). ( C ) Merged confocal images of a single z stack of the boxed region (ii) at a higher magnification: double immunolabeling for GFAP (red) and PLA A2A-D2 heteroreceptor complexes (green). ( D ) Colocalized map of the boxed region (ii); the colocalization analysis plugins—colocalization threshold with GFAP as ROI—was used to create the colocalized map; ImageJ Fiji software. A complete lack of stain for PLA A2A-D2 heteroreceptor complexes was obtained in the negative control experiments, performed avoiding the conjugation of a primary antibody with the Duolink Probes. In the figure the merges of the maximum intensity projections of two representative fields are shown: PLA for A2A-D2. heteroreceptor complexes without the primary anti-A2A antibody ( E ) or the primary anti-D2 antibody ( F ). Scale bar 25 µm or 10 µm

Article Snippet: In situ PLA was performed on 10μm rat striatal slices using the primary antibodies (mouse anti-A2A (1:200, Merck Millipore Corporation, 05-717, Burlington, MA, USA); rabbit anti-D2R (1:200, Alomone Labs, ADR-002, Jerusalem, Israel); goat anti-GFAP (1:500, Santa Cruz Biotechnoloy Inc, Dallas, TX, USA, sc-6170), the Duolink in situ PLA detection kit (DUO92014, Sigma-Aldrich, St Louis, MO, USA) and Alexa Fluor 546-conjugated donkey anti-goat (1:500; Molecular Probes, Eugene, OR, USA).

Techniques: Proximity Ligation Assay, In Situ, Confocal, Immunolabeling, Analysis, Software, Staining, Negative Control, Conjugation Assay

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