12 well plates

MV4-11 cells were seeded in 12-well plates (AGC Techno Glass Co. Ltd., Shizuoka, Japan) at a concentration of 2 × 10⁵ cells/well and cultured overnight. The cells were treated with gilteritinib concentrations of 1, 3, 10, and 30 nM or vehicle (0 nM), and incubated for 24 hours.
The cells were subsequently harvested and fixed in ice-cold 70% ethanol and maintained at 4°C. Following fixation, the cells were washed with phosphate-buffered saline (PBS) and were resuspended in Guava^® Cell Cycle Reagent (Merck Millipore Corporation, Darmstadt, Germany). Cell cycle distribution was measured using a Guava^® PCA microcytometer (Merck Millipore Corporation), and was analyzed in 5000 cells per sample using CytoSoft™ software (Merck Millipore Corporation). The mean percentages of cells in sub-G1, G1, S, and G2/M phases were derived from four independent assays.

Primary cultures of rat cortical neurons were prepared from cerebral cortices of Sprague-Dawley rats at embryonic day 17 [Japan SLC (Shizuoka, Japan)], as described previously [12 (link),13 (link)]. All animal care and experimental protocols were approved by the Animal Experiment Committee of Takasaki University of Health and Welfare (Authorization No. 1733, 1809, 1913, and 2008), and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Takasaki University of Health and Welfare. The cells were seeded (1.8 × 10⁶ cells) and cultured in poly-l-lysine-coated 6-well plates [AGC Techno Glass (Shizuoka, Japan)] for RT-PCR, and 8 × 10⁵ cells were cultured on 18-mm-diameter poly-l-lysine-coated coverslips [Matsunami Glass Ind., Ltd., (Osaka, Japan)] in 12-well plates (AGC Techno Glass) for immunostaining. The cells were cultured using Neurobasal medium [Thermo Fisher Scientific (Waltham, MA, USA)] containing B27 supplement (Thermo Fisher Scientific), 2 μg/mL gentamicin (Thermo Fisher Scientific), and 0.5 mM glutamine (Thermo Fisher Scientific). Half of the culture medium was replaced with fresh medium every 3 days. Each experiment was performed at 13 days in culture.

The C2C12 mouse myoblast cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained using 10‐cm dishes (Corning, One Riverfront Plaza, NY) at 37°C under an atmosphere containing 5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (Sigma‐Aldrich), penicillin (100 U/ml; Nacalai Tesque, Kyoto, Japan), and streptomycin (100 μg/ml; Nacalai Tesque). C2C12 myoblasts were plated in 12‐well plates (AGC TECHNO GLASS, Shizuoka, Japan) and confluent myoblasts were differentiated by incubation in differentiation medium containing 2% horse serum (Sigma‐Aldrich) in DMEM for 5 to 7 days.