Inhibitor screening

Drug screening was performed using SCAD inhibitor kits (kit I, ver. 3.3; kit II, ver. 2.0; kit III, ver. 1.6; kit IV, ver. 2.3) obtained from the Molecular Profiling Committee, Grant-in-Aid for Scientific Research on Innovative Areas “Platform of Advanced Animal Model Support” from the Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI 16H06276). Sora cells were plated at 11,000 cells/well in a 96-well plate, which was determined in a preliminary experiment to ensure logarithmic growth for 72 h. After incubation for 24 h, a total of 331 drugs were added at final concentration of 10 μM. The drugs were dissolved in DMSO at final concentration of 0.001% and same amount of DMSO was added to control well. After 48 h incubation, the cell toxicity was evaluated using a sulforhodamine B (SRB) assay [26]. Briefly, cold 10% trichloroacetic acid solution (Wako) was added to each well, and the plates were incubated at 4°C for 1 h for fixation. Then, the plates were washed four times with slow-running tap water and dried at room temperature. Subsequently, 0.057% SRB solution (Sigma-Aldrich) was added to each well. The plates were incubated at room temperature for 30 min and rinsed four times with 1% acetic acid to remove unbound dye. Tris base solution (10 mM, pH 10.5) was added to each well, and the plates were incubated for 30 min at room temperature. Absorbance at 510 nm was measured using a Bio-Rad Microplate Reader Model 550 (Bio-Rad Laboratories, Hercules, CA, USA).