Celltiter glo 2.0 assay

Cell proliferation assays were performed as previously described^{50 (link)}. Cells were cultured in growth medium at a density of 5 × 10³ cells/well in 96-well plates followed by incubation for 72 h. Adherent cells were then incubated with WST-8 cell counting reagent (Wako Pure Chemical Industries Osaka, Japan) for 2 h. Optical density was measured at 450 nm using a plate reader (Bio-Rad Laboratories, Hercules, CA). ATP assays were used to examine proliferation in spheres using the CellTiter-Glo® 2.0 Assay (Promega, Madison, USA) according to the manufacturer’s protocol.

Breast cancer cells were seeded at a density of 3000 cells per well in 96-well flat-bottomed plates and incubated for 72 h in culture medium. Cells were then treated with ATO, ATRA, or their combination. Control cells received dimethyl sulfoxide (DMSO) at a concentration equal to that of drug-treated cells. At 72 h, cells were counted after trypsin digestion, or medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide was added to each well for a 2 h incubation at 37 °C, followed by removing the media before adding 200 μl DMSO. Absorbance was determined at 570 nm. Leukemia cells were seeded at a density of 5000 cells per well in 96-well flat-bottomed plates, and incubated for 72 h in the culture medium. The number of cells was determined by CellTiter-Glo^® 2.0 Assay (Promega, Madison, WI, USA) following the manufacturer’s instructions.

Intracellular ATP levels were measured using the CellTiter-Glo 2.0 Assay (Promega) according to the manufacturer’s instructions. Briefly, 24 h after the treatment of each drug, or 48 h after the transfection of siANT2 or negative control siRNA in a 96-well opaque-walled plate, CellTiter-Glo Reagent was added to the medium, and cells were lysed on an orbital shaker for 2 min. After an incubation at room temperature for 10 min, luminescent signal intensity was read on a luminometer (Berthold Technologies USA, LLC, Oak Ridge, TN, USA). Obtained data were normalized to the absorbance measured by a Cell Counting Kit-8 assay, which was performed prior to cell lysis. All experiments shown were replicated at least twice.

MOLM-13 and MV4-11 cells were seeded at 10,000 cells/well in 96-well plates. After culturing overnight, cells were treated with gilteritinib, quizartinib, or midostaurin in the presence or absence of FL at 25 ng/mL for 3 days [21 (link), 26 (link)]. FLT3^wt- or FLT3-ITD-expressing Ba/F3 cells were seeded at 1,000 cells/well in 96-well plates and treated with gilteritinib or quizartinib for 3 days in the presence or absence of FL at 25 ng/mL for 3 days. Cell viability was measured using the CellTiter-Glo 2.0 Assay (Promega). Measurement of luciferase activity was performed using the ARVO X3 (PerkinElmer) or SpectraMax Paradigm (Molecular Devices). Data were analyzed using GraphPad PRISM 7 software (GraphPad). Cell viability was calculated by defining the survival of untreated cells and medium control wells as 100% and 0%, respectively.

Cells were seeded in 96-well solid white plates at a density of 2.0 × 10³ cells per well in 80 μL of complete medium and were cultured for 24 h. The cells were treated with various concentrations of eribulin (0.001, 0.1, 0.25, 0.5, 1, 5, 10 nM) for 3 days. An ATP assay was performed according to the manufacturer’s protocol (CellTiter-Glo^® 2.0 assay, Promega). 50 μL of reagent solution was added to each well, and the plates were kept in the dark for 15 min. The absorbance of luminescence was read at 578 nm in a luminometer (SpectraMax^® Paradigm^®, Molecular Devices, CA, USA)^{58 (link)}.