Ingenol

BEAS-2B normal human epithelial cell line was purchased from ATCC (Catalog number: CRL-9609) and cultured according to vendor instructions using BEGM kit from LONZA (Catalog number: CC-3170). Cells were cultured on 96-well black μ-plate from ibidi (Catalog Number: 89626) for imaging studies. Tanespimycin (abcam ab141433), Acetylcysteine (Cayman, 20261), Amifostine (Cayman 14398), Bortezomib (Ayman 10008822), FK-866 (Cayman 13287), Gemcitabine (Cayman 11690), Idarubicin (Cayman 14176), NVP-AUY922 (Cayman 10012698), NVP-BEZ235 (Cayman 10565), PIK-75 (Cayman 10009210), SN-38 (Cayman 15362), Tretinoin (Cayman 11017), YM-155 (Cayman 11490), Ingenol (Cayman 14031), Sulforaphane (LKT S8044), CD-437 (Sigma C5865), and Parbendazole (Sigma 1498706) were dissolved in DMSO. 1000x concentration working solution was used for downstream experimentation.

Human peripheral blood mononuclear cells were obtained from healthy, unidentified blood donors (Gulf Coast Regional Blood Center (GCRBC), TX). Naive CD4 T cells were isolated from PBMCs by negative selection and activated in non-polarizing conditions at 0.5x10⁶ cells/mL in the presence of 2μg/mL αhuman IL-12, 1μg/mL αhuman IL-4, 10ng/mL TGF-β and αCD3/28 stimulation beads at one bead/cell (Dynal/Invitrogen, CA) as previously performed [33 (link), 34 (link)]. Subsequently, cells were expanded with 30 IU/mL hIL-2 in RPMI supplemented with 1% L-Glutamine, 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin. Incubation with FTY720 was performed on cells from various time points of our model for 24–72 hours at concentrations of 30-100nM. HIV-1_NLAD8 and HIV-1_NL4-3 viruses were generated in HEK293FT cells by calcium phosphate transfection and latently infected cells were generated as previously described [33 (link), 34 (link)].
For latency reversal experiments, cells were cultured at 0.5x10⁶ cells/mL in the presence of 30 IU/mL hIL-2. Reactivation conditions included: IL-2 only, 66nM FTY720, or one of the following LRAs +/- 66nM FTY720: αCD3/28 stimulation beads (one bead/cell), SAHA (330nM, Cayman), Pam2CSK4 (1μM, Invivogen), HODHBt (100μM, A.K. Scientific), or Ingenol (100nM, Cayman). Following 48 hours of culture, intracellular p24-gag was assessed by flow cytometry.