Ingenol 3 20 dibenzoate

The natural compound ingenol-3-dodecatrienoates extracted from Euphorbiaceae and the derivate esters Ingenol A, B and C were isolated by Kyolab Laboratories (São Paulo, Brazil). Ingenol 3,20-dibenzoate, PMA, prostatin and the PKC inhibitors Gö 6976, Gö 6983 and Ro-31–8220 were obtained from Santa Cruz Biotechnology. SAHA was obtained from SelleckChem (Houston, USA). All these compounds were diluted to specific concentrations in DMEM or RPMI, and the final concentration of solvent (DMSO) was always less than 1%. We used 20 ng/ml of TNF-α (eBiosciences) as positive control of reactivation. Levels of P-TEFb components were evaluated by immunoblotting using specific antibodies anti-CDK9 and anti-Cyc T1 (Santa Cruz Biotechnologies, cat. number 484 and 10750, respectively). Anti-tubulin (Abcam, cat. number 56676) was used to normalize proteins level.

TNFα (Sigma-Aldrich) was used at 0.5–10 ng/ml. Human αCD3/αCD28 Dynabeads (Invitrogen) were used at a 1 bead/cell ratio. JQ1 (Cayman Chemical) was used at 0.1–10 μM. Ingenol 3,20-dibenzoate (Santa Cruz Biotechnology) was used at 5–200 nM, and SAHA (Merck) was used at 110 nM, 330 nM, or 1 μM. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) was used at 10 nM and ionomycin (Sigma-Aldrich) was used at a concentration of 500 nM. UNC926 (Tocris Bioscience) was used at a concentration of 10 nM–100 μM. AZ505, AZ506, and AZ391 were used at a concentration of 10 nM–10 μM.