Cell viability and proliferation assays

Cells were plated in 96-well plates at 1×10⁴/well density and exposed to drugs in duplicate or triplicate as indicated. Relative cell numbers (compared to vehicle-treated control) were estimated after 48 hours by measuring ATP levels by CellTiter Glo (Promega) for suspension cultures, or by nucleic acids content by methylene blue retention for adherent cultures. As ATP levels as well as DNA and especially RNA content differ between cell lines, normalization to untreated was necessary for adequate comparison. CellTiter Glo assay was performed according to manufacturer’s instructions. For methylene blue staining cells were first thoroughly washed in PBS, then simultaneously fixed and stained by 0.5% methylene blue (Sigma) in 50% methanol, and plates were extensively washed under running water and let to air-dry. Retained methylene blue was solubilized by 0.1% SDS, and OD₅₉₅ was measured by SpectraMax plate reader. Data are representative of at least two independent experiments in each cellular system.

For the ex vivo anthracycline dose-response studies on primary AML samples, 50 nl of each compound were dispensed via acoustic-based transfer into clear 384-well plates dissolved in DMSO at 1000-fold their respective screening concentration. 5000 cells were seeded in each well in 50 μl media on top of these compounds and incubated for 72 hours. ATP levels were measured as surrogate for cell viability (CellTiter Glo, Promega) according to manufacturer’s protocol. Data for each AML sample were normalized to the 32 negative control (DMSO) wells on each plate (100% viability) and the 32 positive control wells (1 μM bortezomib, set to 0% viability) after outlier removal. Clinical characteristics of patient samples are summarized in Supplementary Table 3. Banking and ex vivo drug testing of leukemic patient samples was approved by the ethical committee of the Medical University of Vienna # EK Nr: 2008/2015.