Tk216

Effect of drugs targeting TF activities that were found to be high in E/R+ leukemia was studied in the glucocorticoid-resistant REH cell line. The experiments were performed in three biological replicates. TK216 (ERG/FLI1 inhibitor) was acquired from MedChemExpress and XRP44X (Ras-Net-Elk-3 inhibitor) from Sigma-Aldrich. The drugs were reconstituted in DMSO. MTS assay was used to determine viable cells in proliferation upon drug treatments with increasing concentrations at 72 h time point. REH cells (10,000 cells/well) were seeded with drugs into 96-well plates with a final volume of 100 μl. Following drug treatment, cell proliferation was measured using CellTiter 96® AQ_ueous One Solution (Promega). Twenty microliters of CellTiter 96® AQ_ueous One Solution reagent per well was added, and cells were incubated for 3 h in a humidified (atmosphere 95% air/5% CO₂) incubator at 37 °C. Absorbance was measured at 492 nm by a spectrophotometer (Thermo Scientific, Multiskan Ex). The background signal (no cells) was subtracted, and the average signal from three technical replicate wells was used in calculations. In parallel, cell viability and count were measured based on Trypan blue (Sigma-Aldrich) staining using Cellometer Mini Automated Cell Counter (Nexcelom Bioscience). Relative proliferation and cell amounts were calculated by normalizing to DMSO as a control sample.