Auranofin

JLat 9.2 and TLat 1D5 were diluted to 1 × 10⁶ cells/mL and treated with chemical modulators of latency. PX12, Tiopronin, and Auranofin (redox protein inhibitors) were acquired from Cayman Chemicals. D106 (NSC 155703) was acquired from the National Cancer Institute Developmental Therapeutics Program. PX12 (60 μM), Tiopronin (4 mM), Auranofin (250 nM, 1 μM, or 4 μM), or D106 (10 mM) were added to cells with and without TNF-α (10 ng/mL) for 24 h and analyzed via flow cytometry to determine HIV reactivation. In the case of JLat 9.2, PMA (200 ng/mL) was also used as a latency reversal agent (LRA) to rule out an LRA-specific response to the inhibitors. In MDMs, cells were treated with the same redox protein inhibitors following differentiation and PMA removal from the media (day 0 postdifferentiation). N-acetyl-L-cysteine (NAC; Cayman Chemicals) was added to MLats for 1.5 h before the addition of Auranofin and kept in the media for the duration of treatment (3 h). Cells were analyzed via flow cytometry at different time-points posttreatment.

Three human myeloma cell lines (JJN3, RPMI8226 and U266) were obtained from Dr. Slavica Vuckovic (QIMR Berghofer Medical Research Institute, Brisbane) and have been authenticated by the Griffith University DNA Sequencing Facility (GUDSF) using the STR profiling method (GenePrint® 10 System, Promega). Bortezomib-resistant (BR) myeloma cell lines (RPMI8226-BR and U266-BR) cells were established previously in our lab [35 ,36 (link)]. Human peripheral blood mononuclear cells (PBMCs) were collected and isolated from the whole blood of healthy individuals under the Griffith University human ethical approval number 2014/392. These cells were cultured in RPMI-1640 medium (Gibco) containing 10% (V/V) fetal bovine serum (FBS) (Bovagen), 200 mM l-glutamine with 100 U/ml penicillin and 100 ug/ml streptomycin (Invitrogen). [Au(d2pype)₂]Cl was synthesized by a modification of the published procedure [37 ] using [AuCl(SMe)₂] as precursor and 1,2-bis(di-2-pyridylphosphino)ethane (d2pype) obtained from Strem Chemicals Inc. Auranofin was purchased from the Cayman Chemicals (Michigan, USA), and N-acetylcysteine (NAC), sodium selenite, and buthionine sulfoximine (BSO) were purchased from Sigma Chemicals (NSW, Australia).