Interleukin 3 (il 3)

Bone marrow mononuclear cells isolated from the femurs and tibiae were cultured for two weeks in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and penicillin-streptomycin in the presence of 10 ng/mL recombinant murine IL-3 (PeproTech, Rocky Hill, NJ, USA) and subsequently cultured for two weeks with IL-3 and 10 ng/mL recombinant murine stem cell factor (Peprotech). The 293T cells (Thermo Fisher Scientific) were cultured in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. MEDMC-BRC6 cells [23 (link)] were purchased from RIKEN BRC (Tsukuba, Japan) and cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) containing the following: 15% FBS, ITS liquid media supplement (Sigma-Aldrich, St. Louis, MO, USA), 50 mg/mL ascorbic acid (Sigma-Aldrich), 0.45 mM α-monothioglycerol (Sigma-Aldrich), 3 ng/mL IL-3, 30 ng/mL stem cell factor (SCF), 1% penicillin-streptomycin solution and 2 mM l-glutamine (Nacalai Tesque, Kyoto, Japan).

The IL-3-dependent hematopoietic cell line Ba/F3 was purchased from Riken Cell Bank (Ibaraki, Japan). SUDHL-1 and Ki-JK cells, derived from NPM-ALK-positive ALCL patients, were purchased from Summit Pharmaceuticals International (Tokyo, Japan) and JCRB cell Bank (Osaka, Japan), respectively. SUDHL-1 cells and Ki-JK cells were cultured in RPMI 1640 medium supplemented with 10% FBS (BioWest), 100 units/ml penicillin (Nacalai Tesque), and 100 μg/ml streptomycin (Nacalai Tesque) with or without IL-3 (2 ng/mL) at 37°C and 5% CO₂. Ba/F3 cells were infected with an empty virus (-) and retroviruses expressing NPM-ALK, its kinase dead mutant (K210R), and TEL-JAK2 using RetroNectin according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan). These cells were cultured in RPMI-1640 medium (Nacalai Tesque, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (BioWest, Nuaillé, France), 100 units/ml penicillin (Nacalai Tesque), 100 μg/ml streptomycin (Nacalai Tesque), 2 ng/mL IL-3 (PEPROTECH), and 5 μg/mL puromycin (InVivoGen).

The IL-3-dependent hematopoietic cell lines Ba/F3 were infected with an empty virus (-) and retroviruses expressing NPM-ALK, its kinase dead mutant (K210R), and TEL-JAK2 using RetroNectin according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan). These cells were cultured in RPMI-1640 medium (Nacalai Tesque, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (BioWest, Nuaillé, France), 100 units/ml penicillin (Nacalai Tesque), 100 μg/ml streptomycin (Nacalai Tesque), 2 ng/mL IL-3 (PEPROTECH), and 5 μg/mL puromycin (InVivoGen). SUDHL-1 and Ki-JK cells, derived from NPM-ALK-positive ALCL patients, were cultured in RPMI 1640 medium supplemented with 10% FBS (BioWest), 100 units/ml penicillin (Nacalai Tesque), and 100 μg/ml streptomycin (Nacalai Tesque) with or without IL-3 (2 ng/mL) at 37°C and 5% CO₂.