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: 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