Human cerebral microvascular endothelial cells (HBEC-5i) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured on 0.1% gelatin solution (ATCC)-coated flasks in DMEM:F12 medium (ATCC) supplemented with 10% foetal bovine serum (Gibco/Thermo Fisher, Waltham, MA, USA), 40 μg/mL endothelial growth supplement (ECGS, ATCC), and 1% penicillin-streptomycin (Beyotime, Shanghai) according to the manufacturer's instructions. HBEC-5i cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
Recombinant HIV-1 Tat clade-B protein (amino acids 1 to 86) was purchased from Prospec (Rehovot, Israel). The previous literature indicated that concentrations of Tat in HIV-infected patients could reach the range of 0.5 μg/mL of serum [24 (link)], so this concentration was used in subsequent experiments. Controls consisted of cells treated with 0.02% DMSO or heat-inactivated Tat (1 μg/mL). Before exposure to 1 μg/mL HIV-1 Tat for 12 or 24 h, confluent HBEC-5i cells were pretreated with 5 μmol/L FTS for 3 h and FTS was retained in the serum-free cell culture medium during Tat treatment as previously described [25 (link)].
Recombinant HIV-1 Tat clade-B protein (amino acids 1 to 86) was purchased from Prospec (Rehovot, Israel). The previous literature indicated that concentrations of Tat in HIV-infected patients could reach the range of 0.5 μg/mL of serum [24 (link)], so this concentration was used in subsequent experiments. Controls consisted of cells treated with 0.02% DMSO or heat-inactivated Tat (1 μg/mL). Before exposure to 1 μg/mL HIV-1 Tat for 12 or 24 h, confluent HBEC-5i cells were pretreated with 5 μmol/L FTS for 3 h and FTS was retained in the serum-free cell culture medium during Tat treatment as previously described [25 (link)].