Fig. 6

Cocaine’s Effect on PXR is Specific, Dose-Independent, and Occurs Primarily in the Nucleus. A-D Immunofluorescent microscopy was performed to evaluate A PXR or C CAR following treatment with cocaine (10 μM, right) or vehicle (left) for 24 h. The fluorescent signal for each respective protein that colocalized with DAPI was separated from that which occurred in the cytoplasm to facilitate analysis of PXR and CAR specifically in the nucleus. One paired representative image, out of 20 individual images, are shown. All scale bars = 50 μm. (B, D) Quantification of the nuclear fluorescent signal from (A) PXR and (C) CAR immunofluorescent microscopy was performed for endothelial cells treated with cocaine (10 μM, burgundy) or vehicle (teal) for 24 h. Twenty independent experiments (represented by individual dots) were performed. Estimation plots are shown where the left y-axis denotes relative fluorescent intensity (RFU, pixels) of the nucleus and the right y-axis reflects the effect size (black bar), which is the difference between means of each condition. Data are represented as mean ± standard deviation. ****p < 0.0001. Unpaired T-test was performed. (E) Western blot was performed to evaluate PXR following 24-h treatment with cocaine (0.01–100 μM) or vehicle (0 μM). β-actin was used for protein normalization. One western blot, representative of six independent experiments, is shown (top). The fold change in relative band intensity for PXR/β-actin was determined by densitometry where vehicle treatment was set to 1. Six independent experiments (represented by individual dots) were performed. The fold change in relative band intensity for PXR relative to β-actin is depicted (bottom). Data are represented as mean ± standard deviation. *p < 0.05. One-way ANOVA was performed. F Western blot was performed to evaluate PXR following 0.5–24 h treatment with cocaine (10 μM) or vehicle (0 μM). β-actin was used for protein normalization. One western blot, representative of six independent experiments, is shown (top). The fold change in relative band intensity for PXR/β-actin was determined by densitometry where vehicle treatment was set to 1. Six independent experiments (represented by individual dots) were performed. The fold change in relative band intensity for PXR relative to β-actin is depicted (bottom). Data are represented as mean ± standard deviation. *p < 0.05. **p < 0.01. ***p < 0.001. One-way ANOVA was performed. (G) Western blot was performed to evaluate PXR following 24-h treatment with cocaine (10 μM), its minor metabolite norcocaine (10 μM), its major metabolite benzoylecgonine (10 μM) or vehicle. β-actin was used for protein normalization. One western blot, representative of 9 independent experiments, is shown (top). The fold change in relative band intensity for PXR/β-actin was determined by densitometry where vehicle treatment was set to 1. Nine independent experiments (represented by individual dots) were performed. The fold change in relative band intensity for PXR relative to β-actin is depicted following cocaine (burgundy), norcocaine (yellow), benzoylecgonine (turquoise), or vehicle (teal) treatment (bottom). Data are represented as mean ± standard deviation. ***p < 0.001. One-way ANOVA was performed