Ba f3

Mouse embryo fibroblast cells, NIH 3T3 (ATCC no. CRL‐1658), were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). NIH 3T3 cells were cultivated in Dulbecco's modified Eagle's medium without sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (100 U·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin). A murine pro B cell line, BA/F3, was purchased from Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and a BA/F3 FGFR1c cell line stably transfected with the FGFR1 gene (isoform IIIc) was provided by D. M. Ornitz (Washington University, St Louis, MO, USA). Both, BA/F3 and BA/F3 FGFR1c cells were cultivated in RPMI‐1640 medium (Thermo Fisher Scientific) supplemented with 10% newborn bovine calf serum (Thermo Fisher Scientific), antibiotics (100 U·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin), β‐mercaptoethanol (50 nm) and mouse interleukin 3 (PeproTech, New Jersey, NJ, USA).

A murine pro-B cell line, Ba/F3, was obtained from Riken RBC (Tsukuba, Japan) and cultured in an RPMI 1640 medium (Thermo Fisher, Waltham, MA, USA) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 ng/mL IL-3 at 37 °C, in a humidified atmosphere containing 5% CO₂ and 95% air.
Ba/F3 cells stably expressing Kusabira Orange (KO) were prepared by conventional transfection with electroporation and cloning by limited dilution. Briefly, 40 µg of KO expression plasmids (AM-V0140, MBL, Tokyo, Japan) was mixed with 5 × 10⁶ cells of Ba/F3 cells in 200 µL Optimem (Thermo Fisher), transferred into a 0.2-cm-gap cuvette, and electroporated by delivering one exponential decay pulse of 140 V and 1000 µF, using the Gene Pulser Xcell Electroporation System with the CE Module (Bio-Rad, Hercules, CA, USA). Transfected cells were returned and cultured in RPMI 1640/10% FBS containing 1 ng/mL IL-3 for 18 h, and selected on the culture media supplemented with 1 mg/mL G418 for 1 month. Stably expressing KO cells were established by cloning with limited dilution in a 96-well plate.