Journal: Frontiers in Cellular Neuroscience

Article Title: Differential Regulation of Adhesion and Phagocytosis of Resting and Activated Microglia by Dopamine

doi: 10.3389/fncel.2018.00309

Figure Lengend Snippet: (A) Activation of p38MAPK by DA in resting microglia cannot be blocked with DA receptor antagonists. Resting BV 2 microglia cells were pre-treated with various DA receptor antagonists (2 μM each, specificity of each antagonist was mentioned in section “Materials and Methods”) for 10 min and then treated with 2 μM DA for 30 min. Activation of p38MAPK was measured as fold increase to the control using immunoblotting with the anti-phospho-p38MAPK antibody. Values are means ± standard error of five independent experiments (ANOVA: ∗ P < 0.05). (B) Activation of p38MAPK by DA in primary microglia. Cultured primary microglia cells from mouse brain were pre-treated with spiperone and decynium and treated with 2 μM DA for 30 min. Activation of p38MAPK was assessed by immunoblotting (ANOVA: ∗ P < 0.05, ∗∗ P < 0.01 compared to control). (C) p38MAPK activation by DA can be inhibited by selective DAT blockers (benztropine and vanoxerine; 2 μM each) and PMAT blocker (Decynium; 2 μM). Combination of DAT and PMAT blockers further inhibited the activation of p38MAPK (ANOVA: ∗ P < 0.05, ∗∗ P < 0.01). (D) Inhibitors of monoamine oxidase did not show any significant effect on p38MAPK activation by DA. Neither pargyline nor rasagiline (2 μM) showed a significant effect on p38MAPK activation by DA in resting microglia (ANOVA: ∗∗∗ P < 0.005).

Article Snippet: Rasagiline and DA transporter blockers, Benztropine and Vanoxerine (GBR-12909), were purchased from MedChemExpress (Shanghai, China) and decynium-22, a blocker for plasma membrane monoamine transporter was from Sigma-Aldrich.

Techniques: Activation Assay, Control, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: A study of the dopamine transporter using the TRACT assay, a novel in vitro tool for solute carrier drug discovery

doi: 10.1038/s41598-020-79218-w

Figure Lengend Snippet: Apparent potency values of dopamine (pEC 50 ) and inhibitory potency values of DAT inhibitors (pIC 50 ) on U2OS-mock, U2OS-DAT or JumpIn-DAT (± dox) cells in TRACT experiments using a non-linear regression analysis model with a fixed pseudo-Hill slope of 1. Values are reported as the mean ± SEM of three to nine individual experiments performed in duplicate ( n indicates the number of biological replicates). Significant difference between two mean potency values was determined by unpaired two-tailed Student’s t-test. *p < 0.05 (compared to U2OS-mock); ## p < 0.01 (compared to U2OS-DAT/dopamine); ††† p < 0.001 (compared to JumpIn-DAT (−dox)). Comparison of multiple mean values to vehicle control was done using a one-way ANOVA with Dunnett’s post-hoc test. ‡‡‡ p < 0.001 (compared to JumpIn-DAT (+dox)/dopamine). &&& p < 0.001 (compared to JumpIn-DAT (−dox)).

Article Snippet: GBR12909 dihydrochloride was purchased from Toronto Research Chemicals (North York, Canada).

Techniques: Comparison, Control

Journal: Scientific Reports

Article Title: A study of the dopamine transporter using the TRACT assay, a novel in vitro tool for solute carrier drug discovery

doi: 10.1038/s41598-020-79218-w

Figure Lengend Snippet: Functional characterization of GBR12909 on DAT in U2OS-DAT cells in a TRACT assay. Cells were pretreated with vehicle, 10 µM ( a,b ) or increasing concentrations ( c,d ) of GBR12909. Representative vehicle-corrected xCELLigence traces after stimulation with ( a ) increasing concentrations of dopamine or ( c ) 3.16 µM dopamine. ( b ) Concentration-effect curves of dopamine in U2OS-DAT cells pretreated with vehicle or 10 µM GBR12909 are shown as the net AUC of the first 120 min after stimulation normalized to the cell response of 31.6 µM dopamine. ( d ) Concentration-effect curve of GBR12909 after addition of 3.16 µM dopamine normalized to 10 µM GBR12909. Data are shown as mean ± SEM of three to six separate experiments each performed in duplicate.

Article Snippet: GBR12909 dihydrochloride was purchased from Toronto Research Chemicals (North York, Canada).

Techniques: Functional Assay, Concentration Assay

Journal: Scientific Reports

Article Title: A study of the dopamine transporter using the TRACT assay, a novel in vitro tool for solute carrier drug discovery

doi: 10.1038/s41598-020-79218-w

Figure Lengend Snippet: Representative saturation binding curve of [ 3 H]WIN35,428 to DAT on U2OS-DAT membranes. Total binding (●) and non-specific binding (○) were determined in the absence or presence of 100 μM GBR12909. Specific binding (■) was obtained by linear subtraction of non-specific binding from total binding. Data are shown as the mean of a representative graph of three separate experiments each performed in triplicate.

Article Snippet: GBR12909 dihydrochloride was purchased from Toronto Research Chemicals (North York, Canada).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A study of the dopamine transporter using the TRACT assay, a novel in vitro tool for solute carrier drug discovery

doi: 10.1038/s41598-020-79218-w

Figure Lengend Snippet: Functional characterization of GBR12909 and cocaine on DAT in JumpIn-DAT cells in the presence of 1 µg/ml dox (+dox) in a TRACT assay. Cells were pretreated with vehicle or 10 µM ( a,b ) or increasing concentrations ( c,d ) of either GBR12909 or cocaine. ( a ) Representative vehicle-corrected xCELLigence traces after stimulation with increasing concentrations of dopamine or ( c ) 3.16 µM dopamine. ( b ) Concentration-effect curves of dopamine in dox-treated JumpIn-DAT cells pretreated with vehicle or 10 µM GBR12909 or cocaine are shown as the net AUC of the first 30 min after stimulation normalized to the cell response of 316 µM dopamine. ( d ) Concentration-effect curve of GBR12909 and cocaine after addition of 10 µM dopamine normalized to 10 µM inhibitor. Data are shown as mean ± SEM of four to nine separate experiments each performed in duplicate.

Article Snippet: GBR12909 dihydrochloride was purchased from Toronto Research Chemicals (North York, Canada).

Techniques: Functional Assay, Concentration Assay