Cell lines and cell culture

Ba/F3 (interleukin [IL]-3-dependent murine pro-B) cells engineered to express FLT3-ITD, SYK-TEL, TEL-SYK, FLT3-ITD+SYK-TEL, and FLT3-ITD+TEL-SYK [10] were provided by Dr. Kimberly Stegmaier. Ba/F3 cells were engineered to over-express wt FLT3 as previously described [6].

Human AML-derived, FLT3-ITD-expressing MV4,11 cells were obtained from Dr. Anthony Letai. The human AML-derived, FLT3-ITD-expressing line, MOLM14 [31], was provided to us by Dr. Scott Armstrong, Dana-Farber Cancer Institute (DFCI), Boston, MA.

The human AML-derived, FLT3-ITD-expressing cell line, MOLM-13 (DSMZ (German Resource Centre for Biological Material), was engineered to express luciferase fused to neomycin phosphotransferase (pMMP-LucNeo) by transduction with a VSVG-pseudotyped retrovirus as previously described [32]. Development of PKC412-resistant cells derived from long-term culture or drug-resistant colonies (MOLM13-R-PKC412 (CFU)) was described previously [28].

All cell lines used in this study were cultured with 5% CO₂ at 37°C, at a concentration of 2×10⁵ to 5×10⁵ in RPMI (Mediatech, Inc., Herndon, VA) with 10% fetal bovine serum (FBS) and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. Parental Ba/F3 cells and Ba/F3-wt FLT3 cells were cultured in RPMI with 10% FBS and supplemented with 2% L-glutamine and 1% penicillin/streptomcyin, as well as 20% WEHI (as a source of IL-3).

Human cell lines were submitted for cell line authentication and were authenticated within 6 months of manuscript preparation through cell line short tandem repeat (STR) profiling (DDC Medical, Fairfield, OH and Molecular Diagnostics Laboratory, Dana-Farber Cancer Institute). All cell lines tested matched >80% with lines listed in the ATCC or DSMZ Cell Line Bank STR. All cell lines were confirmed to be virus- and Mycoplasma-free.