Prostratin

The human monocytic cell line U937, CD4⁺ T lymphocytic cell line Jurkat (ATCC, Manassas, VA), the chronically infected U1 and J1.1, and CD4⁺ reporter T‐cell line, CEM‐GFP (AIDS Research and Reference Reagent program, NIH, USA) were grown in RPMI 1640 (Cell Clone), with 10% FBS (MP Biomedicals) and 2 mM l‐glutamine (MP Biomedicals) supplementation. Vs treatment, transfection, and HIV‐1 infection were carried out in Opti MEM media (Hyclone). HIV activation in U1 cells was carried out by treatment with 5 ng/ml of phorbol ester PMA (Sigma) or 1.25 μM prostratin (Sigma).

50,000 J-Lat 10.6 and JNLGFP cells were aliquoted into 96-well plates in 200 μL medium. Each of the following drugs were added to the cells in triplicate at the indicated concentrations: BMH-21 (MedKoo, 406586), RA190 (Calbiochem, 5.30341.0001), ERW1041E (Sigma, 509522), ZDON (Calbiochem, 616467), ZDON (Calbiochem, 15227), tubacin (Sigma, SML0065), b-AP15 (Cayman chemical, 11324; AdooQ Bioscience, A15391; MedKoo, 406452), capzimin (Glix labs, GLXC-09966), thiolutin (MedKoo, 525293; Cayman chemical, 11350), WP1130 (Cayman chemical, 15227), P005091 (MedKoo, 406577; Cayman chemical, 15224), IU1 (AdooQ, A13209; Cayman chemical, 10617), TCID (Cayman chemical, 16353; Sigma, SML1402), acitretin (Sigma, 44707), prostratin (Sigma, P0077), ingenol (Sigma, SML1318), vorinostat (Sigma, SML0061), JQ1 (Sigma, SML1524), I-BET151 (Tocris, 4650), 5-azacytidine (Sigma, A3656), romidepsin (Sigma, SML1175), PR619 (AdooQ Bioscience, A13190). For control groups, 0.1% DMSO was used. After 48 or 72 h of treatment, GFP expression in the cells were measured by flow cytometry. Cell viabilities were determined by forward scatter vs. side scatter gating using untreated cells as the control. The data were analyzed with FlowJo software and plotted as bar graphs.

Carfizomib (S2853), JQ1 (S7110), salubrinal (S2923), PR-957 and bortezomib (S1013) were purchased from Selleck Chemicals (Houston, TX, USA). SAHA, prostratin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol was purchased from MedChem Express (Monmouth Junction, NJ, USA). TNF-α was purchased from R&D Systems. Antibodies specific to HSF1 and β-actin were from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific to PARP1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific to p24 (183-12H-5C) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Plasmid vector LentiCRISPRv2, pCDH, pVSVg and psPAX2 were obtained from Addgene. PolyJet was purchased from SignaGen Laboratories (SL100688, USA). Puromycin was purchased from VWR-AMRESCO (USA).

EIF2α, p-EIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1 (Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C, STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132, BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol, parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt, Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat 10.613 (link), U141 (link) and ACH242 (link) cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO₂. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.

J-Lat C11 cells which were generated and used in our lab [26 (link), 28 (link), 48 (link)–50 (link)], were latently infected Jurkat cells encoded to express green fluorescent protein (GFP) as a marker for Tat-driven HIV long terminal repeats (LTR) expression. J-Lat A10.6 cells were obtained from NIH AIDS Reagent Program. Both the two types of cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% Pen/strep at 37°C under 5 % CO₂ humidified atmosphere. For the reactivation of HIV-1 LTR, cells were treated with bromosporine (Selleckchem), prostratin (Sigma) or TNF-α (Chemicon International).

J-Lat C11 cells^{24 (link),26 (link)} (established in our lab) and A10.6 cells^{29 (link),54 (link)} (obtained from NIH AIDS Reagent Program) harboring latent, transcriptionally competent HIV-1 provirus that encodes GFP as an indicator of viral activation were cultured in RPMI1640 medium (Gorning) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin and 100 μg/ml streptomycin (Gibco) in a 37 °C incubator containing 5% CO₂. For in vitro reactivation experiments, C11 cells or A10.6 cells were stimulated with RVX-208 (Selleckchem), PFI-1 (Selleckchem), JQ1 (Selleckchem), SAHA (Sigma-Aldrich), prostratin (Sigma-Aldrich) or TNF-α (Sigma-Aldrich) respectively and subjected to determine GFP expression using flow cytometry (BD Calibur).