Epigenetic probes

Epigenetic probes (Cayman Chemical, USA) were dissolved in DMSO to a concentration of 20 mM (Table 1). The CF41.Mg cells were seeded at 2000/well in 96 well plates (Corning, USA) containing 100 µl of supplemented media as described. After 24 h, media was replaced by new culture media containing different concentrations of Epigenetic probes, ranging from 10 µM to 0.00064 µM. Epigenetic probes were added in six replicates per concentration. After 72 h, 10 µl of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT - 5 mg/mL) was added to each well and formazan crystals were produced over a 2 h incubation period. One hundred µl of DMSO were added to dissolve crystals. Optical density at 540 nm was measured in a Fluorstar Optima (BMG Labtech, Germany). The concentration of compounds resulting in IC₅₀ was calculated for each cell line using nonlinear regression test performed in GraphPad Prism (version 6.00 for Windows, GraphPad Software, USA).

The epigenetic probes (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethylsulfoxide (DMSO) to a concentration of 20 mM. A549 cells were seeded at 5000/well in 96-well plates (Corning, NY, USA) containing 100 µL of supplemented media, as described previously. After 24 h, the medium was replaced with fresh culture medium containing different concentrations of epigenetic probes, ranging from 10 µM to 13.72 nM. epigenetic probes were added in six replicates per concentration and the experiments were performed in triplicate. After 72 h, 10 µL of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) was added to each well and formazan crystals were produced over a 2 h incubation period. One hundred microliters of DMSO were added to dissolve the crystals. The optical density at 540 nm was measured using Fluorstar Optima (BMG Labtech, Ortenberg, Germany). The concentration of the compound corresponding to the IC₅₀ was calculated using a nonlinear regression test performed in GraphPad Prism (version 6.00 for Windows, GraphPad Software, USA).