Fig. 5.

Cocaine Decreases PXR, But Not CAR, in Endothelial Cells. A Schematic representation depicting the transcriptional activity of PXR and CAR following ligand binding. (B-C) Western blot was performed to evaluate (B) PXR and (C) CAR following 24-h treatment with cocaine (10 μM) or vehicle. β-actin was used for protein normalization. One western blot, representative of 15 independent experiments, is shown. (D-E) The fold change in relative band intensity for (D) PXR/β-actin and (E) CAR/β-actin was determined by densitometry where vehicle treatment (teal) was set to 1 (right). Fifteen independent experiments (represented by individual dots) were performed. Estimation plots are shown where the left y-axis denotes fold change in relative band intensity for the protein of interest relative to β-actin 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. (F-G) Immunofluorescent microscopy was performed to evaluate (F) PXR or (G) CAR (green) following treatment with cocaine (10 μM, right) or vehicle (left) for 24 h. DAPI was used to visualize nucleus (blue). One paired representative image, out of 20 individual images, are shown. All scale bars = 50 μm. (H-I) Quantification of the fluorescent signal from (H) PXR and (I) 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) 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