Kribb11

MCF7 (HTB-22; ATCC), T47D (HTB-133; ATCC), and LCC1, LCC2, and LCC9 (provided by the laboratory of Robert Clarke) cells were grown in DMEM (#11965–092; Gibco) supplemented with 10% fetal bovine serum (#12483020; Invitrogen), 100 μM MEM nonessential amino acids (#25–0250; Cellgro), 2 mM L-glutamine (#25030–081; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (#15170–063; Gibco). Cells were treated with (Z)-4-Hydroxytamoxifen (#H7904; Sigma-Aldrich), Fulvestrant (ICI 182,780, #I4409; Sigma-Aldrich), KRIBB11 (#385570; Sigma-Aldrich), MG-132 (#C2211; Sigma-Aldrich), or vehicles as indicated. For heat shock, the cells were grown at 42°C for the indicated times.

In vitro treatments were performed as described by us earlier [1 (link)]. Briefly, 1 × 10⁶ 4T1 cells were pretreated in cell culture with 5 µM KRIBB11 (#385570, Sigma-Aldrich Co., St. Louis, MO, USA) or 0.01% DMSO (#D2438, Sigma-Aldrich Co., St. Louis, MO, USA) for 1h before mEHT. The cell suspension was transferred into a plastic bag for treatment with the LabEHY 200 in vitro applicator. A thermosensor (TM-200 thermometer, Oncotherm Ltd., Budaörs, Hungary) was inserted into the bag for temperature follow-up. The unit was placed in glass cuvette (filled with distilled water), which was inserted between the two electrodes of the in vitro applicator (Oncotherm Ltd., Budaörs, Hungary). An average of 4 ± 1 Watts was applied with the same amplitude-modulation (AM) as in the in vivo experiments. The temperature rise of the cell suspension was around 2.3 ± 0.8 °C/min. Cells were treated for 30 min in a temperature-driven way to maintain 42 °C in the cell suspension. Cells were collected 2 h after mEHT treatment, lysed with Tri-Reagent (#TR118/200, Molecular Research Center, Inc., Cincinnati, OH, USA) and processed for RNA isolation.

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.

A549 or HeLa cells were seeded in 96-well plates the previous day and infected with modified VACV viruses as specified in the text at an MOI of 1 (fluorescence readout) or 0.1 (viral titer). For drug treatment, compounds were added at specified concentrations prior to virus addition. Inhibitor compounds: Triptolide, Quercetin (Tocris Bioscience), KRIBB11 (EMD Millipore), KRIBB3, Pifithrin-μ, Myricetin (Sigma Aldrich), and Heat Shock Protein Inhibitor I/KNK437 (Santa Cruz Biotechnology, Inc.) For fluorophore assays, cells were fixed at 18 hpi with 4% formaldehyde. Plates were read on a Tecan infinite M1000 for Venus (excitation: 515 nm and emission: 528 nm) and mCherry (excitation: 587 nm and emission: 610 nm). For plaque assays, virus was collected 24 hpi and titered by plaque assay.