Fig 2: Network-based analysis of interaction network corresponding to candidate genes. (A) The node degree distribution of PPI network. The number of genes is plotted as a function of their degree reflecting a power-law like distribution. The red line corresponds to a power-law distribution. (B) Sub-network of candidate genes. The large-sized black-colored nodes represent the candidate genes (ABCC1, ABCG2, CERK, MMP-2, MMP-9, and SPHK1), while the small gray color nodes represent the corresponding interactor genes. Interactions are represented in gray color and the depth of color represents the strength of correlations. (C) Shortest path lengths among candidate genes. Heatmap of shortest path length among the candidate genes, where the values represent the number of shortest paths between any pair.

Fig 3: Correlation of CERK with (A) ABCC1, (B) ABCG2 and SPHK1 with (C) ABCC1, (D) ABCG2 in local cohort and CERK with (E) ABCC1, (F) ABCG2 and SPHK1 with (G) ABCC1, (H) ABCG2 in TCGA cohort.

Fig 4: Cocaine Increases BCRP, MRP4, and P-gp Transport Activity.Endothelial cells were loaded with dyes specific for (A) BCRP (Hoechst 33342, 5 µg/mL), (B) MRP4 (monobromobimane, 10 µM), and (C) P-gp (rhodamine 123, 10 µM) for 1 hour (grey) and the dyes allowed to efflux out of the cell for four hours following pre-treatment with cocaine (10 µM, burgundy) or vehicle (teal). The cells were also pre-treated with specific inhibitors of (A) BCRP (10 µM, fumitremorgin), (B) MRP4 (10 µM, ceefourin 1), (C) P-gp (10 µM, ritonavir) (yellow) or PXR (resveratrol, 10 µM, lavender). Flow cytometric analysis was performed to evaluate the fluorescence and one histogram, representative of five independent experiments is shown (left). The fluorescent signal from flow cytometry was determined for endothelial cells pre-treated with cocaine (10 µM, burgundy) or vehicle (teal). Five independent experiments (represented by individual dots) were performed. Estimation plots are shown where the left y-axis denotes the mean fluorescent intensity (MFI, pixels) for (A) Hoechst 33342 (B) monobromobimane, and (C) rhodamine 123 and the right y-axis reflects the effect size (black bar), which is the difference between means of each condition (right). Data are represented as mean ± standard deviation. *p<0.05. **p<0.01. Paired T-test was performed.

Fig 5: Cocaine Modulates Drug Transporter Expression.Immunofluorescent microscopy was performed to evaluate (A) BCRP, (B) OAT1, (E) ENT1, (F) OAT3, (I) MRP1, (J) OATP1A2, (M) MRP4, (N) OATP2A1, (Q) MRP5, or (R) P-gp (green) following treatment with cocaine (10 µM, right) or vehicle (left) for 24 hours. DAPI was used to visualize nucleus (blue). One paired representative image, out of 20 individual images, are shown. All scale bars = 50 µm. (C, D, G, H, K, L, O, P, S, T) Quantification of the fluorescent signal from immunofluorescent microscopy was performed for endothelial cells treated with cocaine (10 µM, burgundy) or vehicle (teal) for 24 hours. 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<0.05. ***p<0.001. ****p<0.0001. Unpaired T-test was performed.