Flt3 s 18

AML cell lines and primary blasts were cultured as previously described(26 (link)). Molm14 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). Quizartinib and sorafenib (LC labs Woburn, MA) was dissolved in dimethyl sulfoxide (DMSO) at stock concentrations of 10 mM. DEC and AZA were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS) to stock concentrations of 5 mM and 10 mM and stored at − 20°C. Depending on the incubation period for each experiment, quizartinib or sorafenib was added every 5 days, and DEC and AZA were added every 24 hours. L-glutamine and Penicillin/Streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Hydrocortisone was purchased from Sigma-Aldrich (St. Louis, MO, USA).
The following antibodies were used for Western blotting: Anti-phosphotyrosine clone 4G10 (Millipore), FLT3 (S-18, Santa Cruz), and beta-actin (#4967, Cell Signaling Technologies). The following antibodies used for flow cytometry were obtained from BD Pharmingen: CD34-FITC (#555821), CD117-PE (#340529), FITC-Annexin V (#556419), and Propidium Iodide (#556463).

Cells were treated as described, washed in PBS, and lysed in lysis buffer (Cell Signaling, Danvers, MA, USA) supplemented with Complete protease inhibitor (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail-2 (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were fractionated on 4–15% Tris-glycine polyacrylamide gels (Criterion gels, Bio-Rad), transferred to PVDF membranes, and probed with antibodies against FGFR1, pSTAT5, STAT5, pMEK1/2, MEK1/2, pERK1/2, ERK1/2, pS6, and S6 (Cell Signaling, Danvers, MA, USA). Other Antibodies: actin (MAB1501, Millipore), FLT3 (S-18, Santa Cruz Biotechnology, Dallas, TX, USA), FGF2 (SC-79, Santa Cruz Biotechnology), and FL (EP1140Y, AbCam, Cambridge, MA, USA).