Serum plus 2

Bone marrow-derived macrophages (BMM) were isolated from femur exudates and differentiated at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle medium supplemented with 5 mM HEPES, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin/streptomycin, 10% Serum Plus II (Sigma) and containing 30% L929 conditioned medium for 7 days with additional medium added on day 3 or 4. L929 conditioned medium was generated using L929 cells (ATCC Cat no CCL-1, RRID:CVCL_0462) grown to confluency and incubated for 2 additional days before collecting the media. Differentiated BMM were collected by washing with ice-cold PBS containing 1 mM EDTA and resuspended in 10 mL supplemented antibiotic-free medium (without phenol red) containing 5% Serum Plus II and counted. Cells were diluted to 4*10⁵ cells/mL and seeded using the following volumes: 100 μL/well: 96-well plate. 1 mL/well: 24-well plate. For 6-well plates and 6 cm petri dishes, 3–5*10⁶ cells were seeded per well.

The HEK293FT cell line was obtained from Invitrogen and maintained in Dulbecco’s modified Eagle’s medium (Sigma, Cat#D5796-500ML) supplemented with 10% Serum Plus II (Sigma, Cat#14009C-500ML), 2 mM L-glutamine (VWR, Cat#02-0131-0100), 1 mM sodium pyruvate (Corning, Cat#25-000-CI), 10 mM nonessential amino acids (Lonza, Cat#13-114E), and 1% minimal essential medium vitamins (Sigma, Cat#M6895-100mL). The IPPK KO was derived from the HEK293FT cell line as previously described [17 (link)].
The proviral HIV plasmid construct consists of pNL4-3 HIV plus CMV-GFP in place of Nef (kindly obtained from Vineet KewalRamani). This HIV construct has Vif, Vpr and Env deleted and has several restriction sites silently added to the Gag gene for cloning purposes. EIAV proviral constructs consisted of the Gag/Pol expression vector pONY3.1 [40 (link)] and the GFP reporter vector pONY8.0G [43 (link)]. The K282A, Q350A, and K351A versions of pONY3.1 were made by standard cloning methods. The VSV-G construct consists of the coding sequence for VSV-g preceded by the EFa1 promoter [44 (link)] and obtained through the NIH AIDS Research and Reference Reagent Program.

All cells were grown in an atmosphere of 5% carbon dioxide (CO₂) at 37 °C. Unless indicated otherwise base media were supplemented with 10% heat-inactivated fetal bovine serum (Serum Plus II; Sigma-Aldrich) and 1% penicillin/streptomycin and L-Glutamine (Mediatech Inc.). Cells were dissociated with 0.25% (w/v) Trypsin—0.53 mM EDTA solution (Mediatech Inc.). 293T parental and Dicer knock out cells (clone 4–25, provided by Dr. Bryan Cullen, Duke University) (RRID:CVCL_0063) were cultured in DMEM (Cellgro). The HCT116 Ago 1/2/3 k.o. cells [53 (link)] were provided by David Corey (UT Southwestern). HCT116 Dicer k.o. cells were purchased from the Korean Collection for Type Cultures (KCTC, clone #43, cat #HC19023) and cultured in McCoy 5 A medium. Neuroblastoma cell line NB7 [54 (link)] was cultured in RPMI1640. Ago2 ko 293T cells (provided by Dr. Klaas Mulder, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands) and HeLa wt and Ago2 ko cells [55 (link)] (provided by Dr. Sarah Gallois-Montbrun, Université Paris Descartes, Paris, France), were all cultured in DMEM (Cellgro). Lipofectamine RNAiMAX was from ThermoFisher Scientific (#13778150).

HEK293T (ATCC), HeLa-LC3-GFP, and MEF (ATCC) cells were maintained in appropriate volume of Dulbecco’s Modified Eagle Medium supplemented with 10% serum (Serum Plus - II, Sigma), 100 units/mL penicillin and 100 units/mL streptomycin (Corning, VA). Schneider’s Drosophila Line 2 (S2 Schneider) was kindly provided by Henri Jasper, Buck Institute for Research for Aging. S2 cells were grown at 25 °C in Schneider’s Insect Medium (Himedia) that was prepared according to the manufacturer’s instructions.

Stock solutions of ISL (Life Chemicals, Niagara-on-the-Lake, ON, Canada) and TDF (provided by AIDS Reagent Program) were prepared in distilled, deionized H₂O. MT-2 cells [22 (link),23 (link)] were cultured in RPMI 1640 (Mediatech, Inc., Manassas, VA, USA), supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (Thermo Fisher, Waltham, MA, USA). (Mediatech, Inc., Manassas, VA, USA). HEK-293/17 [24 (link)] and TZM-GFP cells from Massimo Pizzato (Trento University, Trento Italy) cells were cultured in DMEM (Mediatech, Inc., Manassas, VA, USA) supplemented with 10% Serum Plus II (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin.