Dovitinib

Human colon cancer cell lines KM12SM, HT29, and HCT116 were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum. The cells were cultured at 100% humidity and 5% CO₂ at 37 °C. The NTRK inhibitors LOXO-101, entrectinib, dovitinib, dovitinib lactate, dovitinib dilactic acid, regorafenib, and crizotinib were purchased from Selleck Chemicals (Houston, TX, USA). We used the annexin V-APC/propidium iodide (PI) apoptosis detection kit (Thermo Fisher Scientific, Rockford, IL, USA).

The human GC cell lines NCI-N87, SNU16, MKN7, MKN28, and AGS were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI-1640 supplemented with 10% fetal bovine serum. The cells were cultured at 37 °C with 100% humidity and 5% CO₂. LOXO-101, entrectinib, dovitinib, dovitinib lactate, dovitinib dilactic acid, regorafenib, cabozantinib, and crizotinib were purchased from Selleck Chemicals (Houston, TX, USA).

PBMCs or RPMI8226 cells (1.0×10⁴ cells/well) were seeded in 96-well plates and incubated for 24 h. After incubation, the cells were treated with dovitinib (Selleck Chemicals, USA) and/or KY-05009 [12 (link)] in complete medium containing 5% FBS for 24-72 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. The absorbance was measured using the Multiscan™ FC microplate photometer (Thermo Fisher Scientific, Boston, MA, USA). Experiments were performed in triplicate.

All cell lines were kept at 37° C and 5% CO₂. 293T cells and JIMT-1 cells were grown in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Gibco). SMF cells were grown in DMEM (Corning) supplemented with 10% FBS and 5 mg/ml insulin (Gemini Bio-Products). All media contained 2 mM glutamine (Corning) and penicillin/streptomycin (Corning), unless stated otherwise. Lapatinib was purchased from Santa Cruz Biotech. Axitinib, Dovitinib, KW-2449, staurosporine, UCN-01, midostaurin, lestaurtinib, PHA-665752, SU-14913, and sunitinib were purchased from Selleck Chemicals.

Lysates were collected in radioimmunoprecipitation assay buffer [RIPA; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritionX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 μg/mL aprotinin]. 25 μg or 30 μg of total protein was separated by 10% or 12.5% SDS-PAGE followed by transfer to Immobilon-P membrane. Immunoblotting reagents were from the following sources: antibodies against p-FGFR (Tyr653/654), FGFR1 (D8E4), FGFR4 (D3B12), p-VEGFR (19A10), VEGFR2 (55B11), p-AKT (D9E), AKT (9272), p-MAPK (D13.14.4E), p44/42 MAPK (Erk1/2), OCT4 (2750), CD133 (D2V8Q), Androgen Receptor (D6F11), and β-actin (4967) antibodies were from Cell Signaling Technology; FGFR2 (C-8), FGFR3 (B-9), and STAT5 (C-17) were from Santa Cruz Biotechnology; ALDH7A1 (CAT:ABO11656) was from Abgent; HRP anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminescence (ECL) reagents were from GE Healthcare. Other reagents included: Dovitinib, BGJ398 and PD166866 were from Selleckchem.