Abt 263

Sorted T cell subsets with latent HIV-1 infection as above were then stimulated with 0.1 μM ingenol-3,20-dibenzonate (IDB, ENZO Life Sciences). ABT-263 (0.2 μM, Adooq Bioscience) and SAR405 (2 μM, MedChemExpress) were added as indicated. After culture for 48 h, the cells were incubated with FITC-DEVD-FMK, followed with APC-annexin V (Biolegend). The cells were then used for intracellular staining of p24 as above and analyzed by flow cytometry. Percentages of cell death were calculated by the loss of viable cells negative for annexin V and DEVD staining: (untreated – treated)/untreated x 100%.
To determine the specificity in the killing of HIV-1 infected cells, uninfected T cell subsets were labeled with CellTrace Violet dye (ThermoFisher Scientific) and mixed with corresponding HIV-1-infected T cell subsets at the ratio of 1:1. The cells were cultured with 0.2 μM BMS-626529, 0.1 μM IDB, 0.2 μM ABT-263 and 2 μM SAR405 as indicated for 48 h. The cells were incubated with DEVD-FMK and annexin V as above and analyzed by flow cytometry.

C4-2, CWR22Rv1, and HEK293T cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and tested for mycoplasma contamination. C4-2 and CWR22Rv1 cells were cultured in RPMI 1640 media, and HEK293T cells were cultured in DMEM media. Both media contained 1% penicillin and streptomycin, as well as 10% fetal bovine serum (FBS). C4-2 EnzR cell lines (EnzR1-C4-2) were generated via chronic culture of CRPC C4-2 cells (EnzS1-C4-2) in media containing increasing Enz concentrations (from 10 µM to 40 µM) for 3 months at each concentration. CWR22Rv1 cells are naturally resistant to Enz and were named as Enz-R3-CWR22Rv1 for these studies. All cells were maintained in a humidified 5% CO₂ environment at 37 °C. All cell lines were authenticated and detected to be mycoplasma and bacteria free following ATCC’s instructions. Enz was used at 10 μM for EnzR1-C4-2 cell culture. ABT263 (Adooq, # A10022, Irvine, CA, USA) was used at 5 μM for EnzS1-C4-2 and EnzR1-C4-2 cells, and at 10 μM for EnzR3-CWR22Rv1 cells. Cycloheximide (CHX) (Adooq, # A10036) was used at 1 μg/mL, MG132 at 10 μM (Adooq, # A11043), Bortezomib at 0.5 μM (Selleckchem, #S1013, Houston, TX, USA), N-acetyl-cysteine (NAC) (Adooq, # A10032) was used at 3 mM, and Z-VAD-FMK at 10 μM (Adooq, # A12373).

AZ’1569 (compound-43), AZ’8037 (compound-25; ref. 11 (link)), AZD1480, and AZD6244 (Selumetinib/ARRY-142866) were obtained from AstraZeneca, 5-fluorouracil (5-FU) and oxaliplatin from the Belfast City Hospital Pharmacy, cetuximab from Merck Serono, and crizotinib from Pfizer. Ruloxitinib, capivasertib, ABT-737, and compound library were purchased from Selleckchem, S6K-18 and ABT-263 from Adooq Biosciences, and SN-38 from Abatra. See Supplementary Materials and Methods for references of non–FDA-approved drugs used. siRNA targeting BCL2L1 and the ON-TARGETplus siRNA library was obtained from Dharmacon. See Supplementary Materials and Methods for details of plasmids.