4.2. Treatments

Doxycycline (#3447, Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 1 µg/mL was added to the culture medium to induce Tat expression in RPMI-8866 cells. After treatment, cells were kept in the Dox-containing medium for 6, 24 and 48 h. To monitor ROS production, Tat expression was induced in the RPMI-8866 cells by treating with 1 µg/mL doxycycline for 6 h (Dox) in the presence or absence of Tempol (#10-2471, Focus Biomolecules, Plymouth Meeting, PA USA) at a final concentration of 80 µM. Rapamycin (#R-0395, Sigma-Aldrich, Saint Louis, MO, USA) was added to culture medium at a final concentration of 200 nM for 6, 24 and 48 h. Purified and functionally active Tat protein was produced by Advance Bioscience Laboratories (ABL, Rockville, MD, USA) and obtained through the NIH AIDS Research and Reference Reagent Program, USA. According to the manufacturer, the Tat protein was >95% pure after purification by heparin affinity chromatography and reverse-phase HPLC with removal of endotoxins. Tat was added to culture medium at final concentration of 250 ng/mL. Cells were kept in the Tat-containing medium for 6, 24 and 48 h.