Fetal bovine serum (fbs)

Human embryonic kidney cells (HEK293T; ATCC) were grown in Dulbecco’s Modified Eagle Medium (Quality Biological, Inc) with 1% L-glutamine (Quality Biological), 1% penicillin- streptomycin (Hyclone) and 10% fetal bovine serum (Quality Biological) at 37 °C in 5% CO₂. HEK293T were transfected with WT-F9- V5-His pcDNA3.1 or CO-F9-V5-His pcDNA3.1 and cultured in medium containing 500 ug/ml G418 to generate stably expressing cell populations. Alternatively, HEK293T cells stably expressing WT or CO FIX were established following transduction with lentiviral vectors, as previously described^{68 (link)}.
An equivalent number of cells were plated in T-flasks and supplemented with 10 ng/ml of Vitamin K3, one day prior to all experiments. The culture medium was replaced with Opti-MEM Reduced Serum Medium (Life Technologies) at approximately 80–90% cell confluency and cells were harvested after an additional 24 hours of incubation. Protein concentration in cell lysates and medium was measured using the Quick Start™ Bradford (Bio-Rad) assay according to manufacturer instructions.

C81 is an HTLV-1-infected T-cell line that expresses Tax protein established from patients with T-cell leukemia. These cells are available through AIDS reagent catalog [53] (link)–[55] (link). Cells were cultured in RPMI-1640 containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine (Quality Biological) and were incubated in a 5% CO₂ incubator at 37°C. Cells were cultured to confluency and pelleted at 4°C for 15 min at 3,000 rpm. The cell pellets were washed twice with 25 ml of phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Quality Biological) and centrifuged once more. Cell pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na₃VO₄, 1 mM DTT, one complete protease cocktail tablet/50 ml) and incubated on ice for 20 min, with a gentle vortexing every 5 min. Cell lysates were transferred to Eppendorf tubes and were centrifuged at 10,000 rpm for 10 min. Supernatants were transferred to a fresh tube where protein concentrations were determined using Bio-Rad protein assay (Bio- Rad, Hercules, CA).

HepG2 cells were obtained (HB-8065, lot: 70015966; American Type Culture Collection, Manassas, VA) and cultured in Eagle’s minimum essential medium with L-glutamate (cat. 50983283; Quality Biological, Gaithersburg, MD), 10% fetal bovine serum (cat. FB12999102; Fisher Scientific, Hanover Park, IL), and 1% penicillin/streptomycin (cat. 15140122; Gibco, Grand island, NY); cells were incubated at 37°C and 5% CO₂. An equal number of cells were seeded in each well of 6-well or 10-cm collagen-treated cell culture plates per treatment group and incubated overnight to attach, with subsequent media changes twice per week. Upon 70%–75% confluency, cells were washed twice with phosphate-buffered saline (PBS) and treated with 100 mmol/L CuSO₄ for 24 hours. Untreated HepG2 control cells were washed twice with PBS and provided with fresh medium. The following day, copper-treated cells were washed twice with PBS and subjected to treatment with PCA (1 mmol/L, cat. P4875; Sigma-Aldrich, St. Louis, MO), 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside in dimethyl sulfoxide (0.1–1 mmol/L, AICAR, cat. 501011663; Fisher Scientific, Hanover Park, IL), or isoproterenol (5–25 μmol/L, cat. I2760; Sigma-Aldrich, St. Louis, MO) for 24 hours.

Jurkat (uninfected T lymphocytes), J1.1 (HIV-1-infected T lymphocytes), U1 (HIV-1-infected promonocytic), and U937 (promonocytic) cells were cultured in complete RPMI 1640 media with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin (Quality Biological, Gaithersburg, MD, USA) and incubated in 5% CO₂ at 37 °C. The cells mentioned above were provided by the National Institutes of Health’s (NIH) AIDS Reagent program. Phorbol 12-Myristate 13-Acetate (PMA; 100 nM and CAT: 16561-29-8, Cayman Chemical, Ann Arbor, MI, USA) was used to differentiate monocytes into monocyte-derived macrophages (MDMs) for 5 days. Cells were treated with TAR-binding molecules, which have been previously described in Abulwerdi et al. [23 (link)] and are shown in Supplementary Figure S1. HEK293T cells (CRL-3216, ATCC, Manassas, VA, USA) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) media.
A set of primary PBMCs (Precision For Medicine, Frederick, MD, USA) were cultured in vitro first in PHA/IL-2 (phytohaemagglutinin/Interleukin-2) for 5 days to obtain activated T cells. On day 5, cells were harvested for the drug-titer assay. On day 7, T cells were then infected with the HIV-1 89.6 strain (MOI: 10) for 3 days, and on day 10, T-cell cultures were then treated with 110FA for 48 h. On day 12, cells were processed for RNA analysis.

THP-1 leukocytes (ATCC, Manassas, (VA,) USA) were maintained at 37 °C in 5% CO₂ in RPMI 1640 medium (ThermoFisher Scientific, Waltham, (MA,) USA), supplemented with 10% fetal bovine serum (FBS, Quality Biological, Gaithersburg, (MD,) USA) and 0.05 mM 2-mercaptoethanol (VWR, Radnor, (PA,) USA). Two Jurkat cell lines, Jn.9 and the CD11a-deficient mutant, J-β₂.7 [66 (link)] (gifts from Dr. Edward Lally, University of Pennsylvania), were cultured in RPMI 1640 medium supplemented with 10% FBS, 0.1 mM MEM non-essential amino acids, 1x MEM vitamin solution, 2 mM L-glutamine, and 50 µg/mL gentamicin [18 (link)].