Facs canto machine

To monitor Hsf1-dependent gene expression GFP was placed under control of the btn2 promoter. To increase reporter sensitivity, the unstable N-terminal domain of Btn2 was fused C-terminally to GFP. GFP fluorescence (excitation: 488 nm, emission: 530 nm) was determined by FACS (Fluorescence Activated Cell Sorting) using the BD FACS Canto machine. Cultures of yeast strains were grown at 25°C or 30°C until log phase (.6–.8 OD₆₀₀). 200 µL of cell culture were added to a flat-bottom 96-well plate with four technical replicates per strain. FACS measured GFP fluorescence of 10.000 cells per strain. The GFP values were normalized by subtracting the values of a negative control.

Single cell suspension was prepared from spleen, liver, and bone marrow and stained with fluorescently labeled antibodies using previously standardized protocol with the antibodies listed in Supplementary Table 3. Intracellular staining was performed following fixation and permeabilization using Fix/Perm transcription factor kit (BD biosciences). Live/dead cell and apoptotic cell discrimination done using 7-AAD and Annexin V staining (BD biosciences) and fixable viability dye 510. Data was acquired using BD FACS Canto machine and analyzed in FLOW JO software.

For the analysis of NKG2D ligand expression, cells were washed twice and incubated with 5mM EDTA in PBS for 5 min to detach the cells. Spheroids were also treated with 5mM EDTA and manually disrupted to achieve a single cell suspension. Cells were then resuspended in PBS supplemented with 0.03% azide with 5% BSA for blocking. Staining was performed in the same buffer using monoclonal antibodies against MICA (2C10, Santa Cruz, Santa Cruz, CA, USA), MICB (mAb 1599, R&D System, Minneapolis), ULBP1 (mAB 170818, R&D System, Minneapolis), ULBP2 (MAB1298, R&D System, Minneapolis), ULBP3 (Clone 166510, R&D System, Minneapolis), HLA-A, B, C (W6/32, Thermo Fisher, UK), or IgG1, isotype control monoclonal antibodies (eBiosciences, San Diego, CA, USA). Cells were then washed twice and labeled with FITC-labeled polyclonal rabbit anti-mouse IgG (STAR9B, Serotec, Raleigh, NC, USA) or goat anti-mouse IgG Cy5 conjugated (AP124S, Chemicon). Propidium iodide (Sigma) was added at a concentration of 50 µg/ml to identify viable cells. Flow cytometry was performed using a BD^®FACSCanto machine and FACSDiva Software (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star).

The cervical cancer cell line HeLa Kyoto, a gift from Heinrich Leonhardt (LMU Munich, Munich, Germany), was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), in a humidified atmosphere, at 37 °C and 5% CO₂. HeLa Kyoto cells stably expressing enhanced green fluorescence protein (GFP), GFP-tagged H2A or GFP-tagged histone variants (H2A.X, H2A.Bbd and H2A.Z.1) were cultured as described [16 (link),19 (link)]. Transient transfections of HeLa Kyoto cells with GFP, GFP–JAZF1 or GFP–Tip60 were performed by using X-tremeGENE HP (Roche, Basel, Switzerland) or FuGENE HD (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Two days after transfection, cells were harvested for several experimental applications. Expression levels of stably or transiently transfected HeLa Kyoto cells were quantified by using a FACSCanto machine (Becton Dickinson, Franklin Lakes, NJ, USA) or a BD Accuri C6 Plus Flow Cytometer (Becton Dickinson). Cells were routinely tested for mycoplasma contamination.