Journal: ACS pharmacology & translational science

Article Title: All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

doi: 10.1021/acsptsci.4c00510

Figure Lengend Snippet: Figure 2. BBB cells differentially express TFV and FTC transporters and nucleotide-metabolizing kinases. Primary human BBB cell monoculture and hepatocyte lysates were processed for LC−MS/MS-based proteomics and protein concentrations for (A) ENT1, (B) MRP4, and (C) MRP1 TFV/FTC transporters, (D) AK2, (E) CKB, (F) PKLR, and (G) PKM TFV nucleotide-metabolizing kinases, as well as (H) CMPK1, (I) DCK, (J) PGK1, and (K) TK1 FTC nucleotide-metabolizing kinases, each with respective protein Western blots to confirm protein abundance in BBB cells. Concentrations of proteins of interest were measured by Proteomic Ruler. Four to five independent experiments with two LC−MS/MS injection replicates each were performed (represented as individual dots). Replicates with missing LC−MS/MS values were omitted from the plot. >2 independent experiments with missing values were reported as not reliably detected (ND) by proteomics analyses. Data represented as mean ± standard deviation. Statistical analysis was performed using Brown−Forsythe and Welch ANOVA or Kruskal−Wallis ANOVA by GraphPad software. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blots were performed as previously described.16 Blots were probed with antibodies with specificity to BCRP (NBP2-22124, Novus Bio, Centennial, CO), P-gp (PA5-61300, Invitrogen), MRP4 (12705, Cell Signaling Technology), MRP1 (Ab24102, Abcam), ENT1 (PA5-116451, Invitrogen), PGK1 (PA528612, Invitrogen), AK2 (11014-1-AP, Proteintech, Rosemont, IL), CKB/CKM (15137-1-AP, Proteintech), TK (15691-1-AP, Proteintech), CMPK (11360-1-AP, Proteintech), DCK (17758-1-AP, Proteintech), PKM1 (15821-1-AP, Proteintech), and PKLR (2456-1-AP, Proteintech), overnight at 4 °C, washed with TBS-T, and probed with the appropriate secondary antibody (ab97023 and Ab97051, Abcam) for 1 h at room temperature.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Quantitative Proteomics, Injection, Standard Deviation, Software

Journal: ACS pharmacology & translational science

Article Title: All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

doi: 10.1021/acsptsci.4c00510

Figure Lengend Snippet: Figure 4. BBB TFV/FTC nucleotide-metabolizing kinases and transporters are impacted in a cell-dependent manner by ART and HIV. Primary human pericyte and astrocyte monocultures were exposed to ART (10 μM TFV, 10 μM FTC, and 10 μM DTG), HIV (5 ng/mL), or HIV + ART (10 μM TFV, 10 μM FTC, 10 μM DTG, and 5 ng/mL HIV) for 24 h at 37 °C, 5% CO2. Treatment with a vehicle was used as a control. Pericytes and astrocytes were lysed and processed for proteomics analyses. Concentrations of proteins of interest were measured by Proteomic Ruler. After exposure, changes in protein concentration were assessed in (A−G) astrocytes (astro) for (A) AK2, (B) CMPK1, (C) ENT1, (D) P-gp, (E) PGK1, (F) MRP4, and (G) MRP1. Similarly, changes in protein concentration were assessed in (H−L) pericytes (per) for (H) CKB, (I) MRP4, (J) MRP1, (K) PGK1, and (L) TK1. The significant changes (A−L) in the concentrations of TFV and FTC transporters and metabolizing enzymes after ART, HIV, or HIV + ART exposure relative to vehicle were (M) summarized for astrocytes (astro) and pericytes (per). An arrow next to each protein denotes an increase (upward arrow) or decrease (downward arrow) in protein concentration after ART, HIV, or HIV + ART exposure relative to the vehicle. Four independent experiments with two LC−MS/MS injection replicates each were performed per condition (represented as individual dots). Replicates with missing LC−MS/MS values were omitted from the plot. >2 independent experiments with missing values were reported as not reliably detected (ND) by proteomics analyses. Data represented as mean ± standard deviation. Statistical analysis was performed using Brown−Forsythe and Welch ANOVA or Kruskal−Wallis ANOVA by GraphPad software. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blots were performed as previously described.16 Blots were probed with antibodies with specificity to BCRP (NBP2-22124, Novus Bio, Centennial, CO), P-gp (PA5-61300, Invitrogen), MRP4 (12705, Cell Signaling Technology), MRP1 (Ab24102, Abcam), ENT1 (PA5-116451, Invitrogen), PGK1 (PA528612, Invitrogen), AK2 (11014-1-AP, Proteintech, Rosemont, IL), CKB/CKM (15137-1-AP, Proteintech), TK (15691-1-AP, Proteintech), CMPK (11360-1-AP, Proteintech), DCK (17758-1-AP, Proteintech), PKM1 (15821-1-AP, Proteintech), and PKLR (2456-1-AP, Proteintech), overnight at 4 °C, washed with TBS-T, and probed with the appropriate secondary antibody (ab97023 and Ab97051, Abcam) for 1 h at room temperature.

Techniques: Control, Protein Concentration, Liquid Chromatography with Mass Spectroscopy, Injection, Standard Deviation, Software