Bryostatin 1

Isolated CD4⁺ T cells were stimulated during 22h with latency reversal agents (LRAs) at the following concentrations: 40 nM Romidepsin (Selleckchem), 30 nM Panobinostat (Selleckchem), 1 μM JQ1 (Sigma-Aldrich), 100 nM Ingenol-3-angelate (Sigma-Aldrich), 10 nM Bryostatin-1 (Tocris Bioscience), the positive control (PMA 81 nM plus Ionomycin 1 μM, both from Abcam), or the negative control (media alone, R10). Drugs were used at concentrations previously shown to be effective at reversing latency in studies performed in CD4⁺ T cells from HIV-infected individuals as well as studies performed in latency models in vitro [23 (link),32 (link),33 (link)]. All compounds were reconstituted in DMSO at the maximum concentration of 0.006%. Moreover, in order to prevent cell death induced by the reactivation of HIV and to evaluate the reactivation effect without confounding variables, cells were pre-treated with a pan-caspase inhibitor named Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone, Selleckchem) [51 (link),52 (link)]. Q-VD-OPh is a potent inhibitor for caspases 1, 3, 8 and 9, which are involved in the intrinsic and extrinsic apoptotic pathways, inhibiting consequently the specific cell death induced by HIV [53 (link)–56 (link)]. In all experiments, cells were treated with 10 μM of Q-VD-OPh for at least 2h prior to the addition of the latency reversal agents.

[γ-³²P]ATP was purchased from PerkinElmer. Phorbol 12-myristate 13-acetate (PMA) (#P8139), was purchased from Sigma–Aldrich. PKC-specific inhibitors GÖ6983 (#S2911), CRT0066051 (#S3422) and LXS-196 (#S6723) were purchased from Selleckchem. CHAPS hydrate (#C5070) and Digitonin (#D141) were purchased from Sigma–Aldrich. L-α-Phosphatidylserine (#840032C) and L-α-Diacylglyerol (#800815C) were purchased from Avanti Polar Lipids, Inc. Lambda Protein Phosphatase (#P0753S) was purchased from New England Biolabs. Trypsin/Lys-C mix (#V5073), Chymotrypsin (#V1062) and Asp-N (#V1621) were purchased from Promega. Bryostatin-1 (#2383) was purchased from TOCRIS. Recombinant Rab7A protein was expressed and purified as previously described [8 (link)]. Recombinant insect cell expressed PKC isoforms namely His-PKCα (DU5084), His-PKCβ (DU33630), Glutathione-S-transferase -PKCγ (DU30188) His-PKCε (DU33642), His-PKCθ (DU29920) and PKCζ (DU1447) were obtained from MRC Reagents and Services (https://mrcintranet.lifesci.dundee.ac.uk).

Drugs, ligands and inhibitors used in this study include etoposide (Sigma Aldrich, St. Louis, MO, USA), SN-38 (Cayman Chemical, Ann Arbor, MI, USA), gemcitabine (Sigma Aldrich), carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma Aldrich), phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich), bryostatin 1 (Tocris, Bristol, UK), Ro 31-8220 (Tocris), TPCA-1 (Sigma Aldrich), recombinant human IL-4 (PharMingen, Franklin Lakes, NJ, USA) and paclitaxel (LC Labs, Woburn, MA, USA). All drugs and inhibitors were dissolved in DMSO (Fisher, Waltham, MA, USA) except recombinant human IL-4 which was dissolved in water and paclitaxel which was dissolved 50:50 Kolliphor/ethanol.