Clone e6 1

The TZM-bl reporter cell line was obtained from the National Institutes of Health (NIH) AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. Jurkat and Clone E6 1 were purchased from ATCC (TIB-152™) Jurkat 2D10 reporter cell line is described previously21 (link). TZM-bl cells were cultured in DMEM high glucose complemented with 10% FBS and gentamicin (10 ug/ml). Jurkat and Jurkat 2D10 cells were cultured in RPMI medium containing 10% FBS and gentamicin (10 ug/ml). Primary culture of human astrocytes and microglia were obtained from the Tissue Culture Core facility of the Comprehensive NeuroAIDS Center (CNAC) in the Department of Neuroscience at the Lewis Katz School of Medicine at Temple University in Philadelphia. In transfection experiments 2D10 cell encompassing LTR_(−80/+66)-Cas9 were electroporated using Neon system (Invitrogen) with the following plasmids: control pKLV-gRNA-empty (6 ug) or pKLV-gRNA LTR A and B (3 ug of each) alone or together with 0 ug, 1 ug, 2 ug, 6 ug of pCMV-Tat86. Total amount of plasmid DNA used in transfections was kept equal (12 ug) with empty pCMV vector (pcDNA3.1). Electroporation conditions: 4 × 10⁶ cells/80 ul of buffer T/12 ug total plasmid DNA, 3 times 10 ms/1350 V impulse using 10 ul tips.

Human Jurkat T cell line (E6-1 clone from American Type Culture Collection) was cultured in RPMI medium supplemented with l-glutamine, charcoal-stripped fetal bovine serum (FBS) at a final concentration of 10%, and penicillin/streptomycin at a final concentration of 1%. Cells were cultured at 37 °C in an air atmosphere containing 5% CO₂. When required for colorimetric assays, RPMI medium without phenol red was used. Cells were maintained at a concentration between 1 × 10⁵ and 1 × 10⁶ viable cells per mL, and fresh medium was added every 2 to 3 days. Experiments were performed in triplicate.

The human Jurkat T cell line (E6-1 clone) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The Jurkat variant with a deficiency of Nck1 (N1KO) was generated using the CRISPR/Cas9 system, as previously described [46 (link)]. Cells were maintained in complete RPMI 1640 medium containing 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, and 1% GlutaMAX (all from Gibco, MA, USA) at 37 °C in a humidified chamber with 5% CO₂.