Formaldehyde

For cell-cycle analysis, drug treated JLat cells were harvested, counted, and washed twice in PBS. Cells were resuspended in flow cytometry staining buffer (R&D Systems, MN, USA) at a concentration of % ≤5 × 10⁵ cells/ml and fixed with 3.9% Formaldehyde (Miltenyi Biotec Inc., CA, USA) for 10 min at RT. Cells were centrifugated at 500 x g for 5 min at 4°C, permeabilized by adding ice cold permeabilization buffer A (Miltenyi Biotec Inc., CA, USA) and incubated on ice for 30 min. After two washing steps with PBS, cells were stained with NUCLEAR-ID^® Red DNA Stain (ENZO Life Sciences Inc., NY, USA) using a 1:1,000-fold dilution. Stained cells were incubated at RT for 30 min. At least 3k reactivated stained cells were collected and analyzed with a BD LSR Fortessa flow cytometer. Cell-cycle distributions are shown as the percentage of cells containing G1, S, and G2 DNA by PE-Cy5-A staining analyzed by FCS Express cell-cycle analysis software (De Novo Software, CA, USA).

JLat 9.2 was treated for 14h with 10ng/ml TNF in combination with either 2.5μM SAHA or 3μM Prostratin. Cells were washed once with 200μl PBS and fixed with 3.9% Formaldehyde (Miltenyi Biotec Inc., CA, USA) for 10 min at room temperature (RT). Afterward, cells were centrifugated at 500 x g for 5 min and 4°C and permeabilized for 30 min on ice using ice cold permeabilization buffer A (Miltenyi Biotec Inc., CA, USA). Two additional washing steps with PBS were performed after cell permeabilization, and cell nuclei were then stained with 0.1mg/ml DAPI (Sigma-Aldrich, St. Louis, USA) for 5 min in the dark at RT. After two washes with PBS, 1.5 × 105 cells were seeded into each experimental well of a half-area glass bottom 96-well plate (Corning) and imaged by taking 36 images per well using a 40x objective.