Additional file 1 of Cocaine regulates antiretroviral therapy CNS access through pregnane-x receptor-mediated drug transporter and metabolizing enzyme modulation at the blood brain barrier
Creators: name: Colón Ortiz, Rodnie
name: Knerler, Stephen
name: Fridman, Lisa B.
name: Mercado, Alicia
name: Price, Amira-Storm
name: Rosado-Franco, Jose J.
name: Wilkins, Hannah
name: Flores, Bianca R.
name: Orsburn, Benjamin C.
name: Williams, Dionna W.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Pharmacology
subject: Biotechnology
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. Brain Microvascular Endothelial Cells and Astrocytes Express Characteristic Markers. Immunofluorescent microscopy was performed to evaluate expression of anticipated markers in (A-G) primary human brain microvascular endothelial cells and (H) primary human astrocytes. (Left panels) Antibodies with specificity to (A) CD71, (B) claudin-5, (C) GLUT-1, (D) VE-Cadherin, (E) occludin, (F) PECAM-1, (G) Zo-1, and (H) GFAP were coupled to Alexa Fluor 488 for analysis. (Middle panels) DAPI was used to visualize nucleus. (Right panels) Merge depicts the combined signal for proteins of interest (green) and DAPI (blue). Representative images, out of 20 independent images, are shown. All scale bars = 50 μm. Figure S2. In vitro Model of the Human BBB. (A) Schematic representation of our transwell BBB model where primary human brain microvascular endothelial cells are seeded on the upper, apical compartment and primary human astrocytes are seeded on the underside of a polycarbonate membrane with 3 μm pores in the basolateral compartment. (B) Schematic representation of albumin permeability assay, where EBA dye is added to the apical portion and permitted to pass to the basolateral side for 30 min at 37 °C, 5% CO2. The media in the basolateral side is collected and spectrophometrically read at OD620 to evaluate BBB permeability. (C-F) The polycarbonate membrane from the BBB model was collected, immunostained, and immunofluorescent microscopy performed. (C-D) Wheat germ agglutinin (WGA) depicts cell morphology in red and (E–F) demonstrates expression of the astrocyte and endothelial cell markers GFAP and VE-Cadherin, respectively in green. DAPI was used to visualize nucleus (blue). Representative images, out of 3–5 independent images, are shown. All scale bars = 50 μm. Figure S3. Cocaine and LPS Do Not Promote Loss of Endothelial Cell Viability. Endothelial cells were treated with cocaine (10 μM, burgundy), LPS (10 ng/mL, fuschia), or vehicle (teal) for (A) 0.5 or (B) 24 h, after which time the cells were loaded with the BD Horizon Fixable Viability Stain 520 for 15 min at 37 °C, 5% CO2. Following treatment, the cells were acquired on the BD LSRFortessa cytometer and Diva software version 9 on the Windows 10 platform (BD Biosciences). Flow cytometric data were analyzed using FlowJo version 10.9 (FlowJo, Ashland, OR). Figure S4. Complete Duration of CYP3A4 Metabolic Activity Assay. Endothelial cells were pre-treated with cocaine (10 μM, burgundy), rifampicin (1 μM, yellow), resveratrol (10 μM, lavender), or vehicle (teal) for 24 h, after which time the cells were loaded with BFC (2 μM). The enzymatic capacity of CYP3A4 to convert BFC to HFC was determined for 80 min as determined by fluorometric quantitation at excitation and emission wavelengths of 405/535 nm. Twelve independent experiments that contained eight technical replicates per condition were performed. Data are represented as mean ± standard deviation. Figure S5. Cocaine Modulates AK1, AK5, and AK6 Expression. Immunofluorescent microscopy was performed to evaluate (A) AK1, (B) AK2, (C) AK5, and (G) AK6 (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. Quantification of the fluorescent signal from 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 &lt; 0.05. **p &lt; 0.01. Unpaired T-test was performed.