Jurkat t cells

Jurkat T cells (ATCC, Shanghai, China) were maintained in RPMI 1640 (Gibco-BRL, Basel, Switzerland) containing antibiotics and 10% FCS (Gibco-BRL). Human embryonic kidney (HEK)-293 T adherent cells (ATCC, Shanghai, China) were cultured in DMEM (Gibco-BRL) supplemented with antibiotics and 10% FCS (Gibco-BRL).

Human embryonic kidney cells (HEK 293T) and Jurkat T cells were purchased from ATCC. The HTLV-1-transformed cell lines MT-2, HUT-102, C8166 and MT-4 were described previously [65] (link), [66] (link). ED40515(-), MT-1, and TL-OM1 cells are clones of leukemic cells derived from ATL patients, kindly provided by Dr. Michiyuki Maeda (Kyoto University). ATL43T is a Tax-negative ATL cell line that was previously described [67] (link). Jurkat Tax Tet-On cells were kindly provided by Dr. Warner Greene [45] (link). 293T cells were cultured in Dulbecco's Modified Eagle's medium (DMEM); Jurkat, MT-2, C8166, MT-4, ATL-43T, HUT-102, ED40515(-), MT-1 and TL-OM1 cells were cultured in RPMI medium. Media was supplemented with fetal bovine serum (FBS; 10%) and penicillin-streptomycin (1×). MISSION shRNAs targeting human IL-17RB, IL-17RA, IL-25, IL-9, Act1, CD40, OX40 and control scrambled shRNA were purchased from Sigma. TRAF6, Tax, IKKα and IKKβ shRNAs were cloned into pYNC352/puro. Target sequences for these shRNAs are listed in Table S3. Tax WT, M22 and M47 were cloned in the pDUET lentiviral vector. Expression vectors encoding κB Luciferase (Luc), pU3R-Luc, pRL-TK (thymidine kinase) have all been described previously [68] (link). Recombinant human IL-9 was purchased from R&D Systems. The IKKβ inhibitor SC-514 was from EMD Millipore.

293 T and 293FT cells (R700-07) were purchased from Invitrogen, and Jurkat T cells were purchased from ATCC. The medium used for these cell lines was Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Transfections were carried out using Lipofectamine 2000 (Invitrogen), except for Jurkat T cells, which underwent electroporation with the Amaxa kit according to the manufacturer’s instructions. The luciferase assay was performed using the luciferase reporter gene assay kit (Promega) according to the manufacturer’s instructions. One day before transfection, 5 × 10⁵ cells were seeded in 6-well culture plates, and the assay was performed after 30 h of transfection.

This research utilized various cell lines including HEK293T cells (ATTC No. CRL-3216), Jurkat T cells (ATCC No. TIB-152), and Magi-5 cells (HeLa cells expressing CD4, CXCR4, and CCR5, and containing the β-galactosidase gene controlled by HIV-1 LTR), which were obtained through the NIH
AIDS Reagent Program. Human primary CD4⁺ T cells were isolated from peripheral blood mononuclear cells of healthy adult HIV-1(−) donors. Human primary CD4⁺ T cells were purified by negative selection using a magnetic-activated cell sorting system (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the protocols from elsewhere [28 (link)]. HIV-1 B subtype NL4-3 viruses were generated by transfecting pNL4-3 in HEK293T cells while CRF07_BC viruses were obtained by clinical isolation or transfecting the infectious clones generated in this study. HIV-1 virions were purified by sucrose gradients according to previously described procedures [49 (link),50 (link)].

In this study, Jurkat T cells (ATCC, Manassas, VA, USA) and Raji B cells (AddexBio, San Diego, CA, USA) were used. Jurkat T cells are cells derived from acute human (male) T cell leukemia and Raji B cells are lymphoblast-like cells established from a male Burkitt’s lymphoma. Cells were cultured, as recommended, in Roswell Park Memorial Institute (RPMI) 1640 medium containing 4.5 g/L D-glucose (hyperglycemic amount), 2.383 g/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.3 g/L L-glutamine, 1.5 g/L sodium bicarbonate, and 0.11 g/L sodium pyruvate (Gibco, Fisher Scientific, Waltham, MA, USA). HEPES buffer was required to maintain the physiological pH while preparing the cells in the device and during live imaging. The media was supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Biosera, Nuaille, France). The cells were incubated at 37 °C and 5% CO₂.