Table 1.
Dopamine receptor subtype agonists and antagonists used in this study.
Table 2.
RT-qPCR primers used for mRNA expression analysis in dHL-60 cells treated with aripiprazole.
Fig 1.
Aripiprazole, but not clozapine, olanzapine, or quetiapine cause loss of dHL-60 cell viability.
dHL-60 cells were treated with 1 μM, 20 μM, or 50 μM in media containing 10% or 2% serum for 48 hours. Cell viability was measured by absorbance of XTT at 450 nm. The average absorbances relative to DMSO control were graphed from at least three replicates. Error bars indicate the standard error of the mean. * indicates statistical significance via ANOVA with Tukey’s post-hoc test.
Fig 2.
Aripiprazole exhibits a dose-dependent increase in dHL-60 cell apoptosis.
Flow cytometry analysis of annexin V and propidium iodide staining in dHL-60 cells treated with 1 μM, 10 μM, or 20 μM aripiprazole for 24 hours in media containing 10% serum. A) Scatterplot of viable cells (lower left quadrant), early apoptotic cells (lower right quadrant), late apoptotic cells (upper right quadrant), and necrotic cells (upper left quadrant) from one representative replicate. At acquisition, 10,000 total cells were measured for each replicate. B) Graph representing the average percentage of early and late apoptotic cells from three independent biological replicates. Error bars indicate the standard error of the mean. * indicates statistical significance via ANOVA with Tukey’s post-hoc test.
Fig 3.
Aripiprazole induces an increase in pro-apoptotic genes in dHL-60 cells.
RT-qPCR analysis of BAK1, caspase-3, and BCL10 mRNA expression in dHL-60 cells treated with 1 μM, 10 μM, or 20 μM aripiprazole for 24 hours. mRNA fold change was calculated using the 2-ΔΔCq method. β-actin and RPL13a were used as reference genes. Data was quantified as described in Taylor et al. (2019). The mean Cq. values were normalized to the reference genes and graphed relative to DMSO controls. The data represent the mean + /- standard error of the mean from three independent replicates. * indicates statistical significance via ANOVA with Tukey’s post-hoc test.
Fig 4.
dHL-60 cells express dopamine receptors D3R and D5R, but not D1R, D2R, or D4R.
Flow cytometry analysis of dopamine receptor surface expression on dHL-60 cells using polyclonal antibodies directed against extracellular epitopes of specific dopamine receptor subtypes. Representative histograms from three independent replicates illustrate the fluorescence intensity of unstained negative controls (black line), secondary antibody controls (blue line), IgG isotype controls (green line), and dopamine-specific receptors (red line).
Fig 5.
Dopamine receptor antagonists fail to inhibit aripiprazole-mediated loss of cell viability.
dHL-60 cells were treated with 20 μM of the dopamine receptor antagonist olanzapine, flupentixol, or eticlopride in the absence or presence of aripiprazole for 48 hours in media containing 2% serum. The average absorbance relative to DMSO control was graphed from at least three replicates. Error bars indicate the standard error of the mean. * indicates statistical significance via ANOVA with Tukey’s post-hoc test.
Fig 6.
Dopamine receptor agonists and antagonists do not affect dHL-60 cells and aripiprazole-induced loss of cell viability.
A) dHL-60 cells were treated with 20 μM of dopamine receptor agonists or B) antagonists in the absence or presence of aripiprazole for 48 hours in media containing 2% serum. The average absorbance relative to DMSO control was graphed from at least three replicates. Error bars indicate the standard error of the mean. * indicates statistical significance via ANOVA with Tukey’s post-hoc test.
Fig 7.
Aripiprazole significantly decreases phosphorylated Src (Y416) levels in dHL-60 cells.
A) Western blot analysis of phosphorylated Src family proteins (Y416) and β-actin in dHL-60 cells treated with 20 μM aripiprazole for 12 hours in media containing 2% serum. B) Quantification of the average fluorescence intensity ratio of phosphorylated Src family proteins and β-actin relative to DMSO control from three biological replicates. * indicates statistical significance via t-test.