Fig. 2

Ba/F3 cell lines harboring secondary mutations along with the EGFR L858R mutation exhibited IL-3-independent growth. a Crystal structure of EGFR. This figure was drawn using the PyMOL Molecular Graphics System based on crystal structure information from PDB ID 2ITZ (EGFR L858R mutation in complex with gefitinib). L747 is located at the start of the loop between the h3 strand and the α-C-helix, D761 is located in the α-C-helix, and T854 is located in the activation loop of EGFR. These residual positions (L747, D761, and T854) are close to the binding sites of ATP or reversible EGFR-TKIs. The secondary mutations were introduced into EGFR along with the L858R mutation. The mutations were confirmed using direct sequencing. b Expression of EGFR in the transfectant Ba/F3 cell lines. The expression of EGFR was confirmed using western blotting. The phosphorylation levels of EGFR were also elevated, similar to that in cells with the L858R mutation alone. β-actin was used as an internal control. c Ba/F3 assay. The cellular growth of Ba/F3 transfectant cell lines grown in the absence of IL-3 were evaluated using an MTT assay. The Ba/F3 and Ba/F3-EGFP cell lines could not grow without IL-3, while the other cell lines (L858R, L858R/L747S, L858R/D761Y, L858R/T854A, and L858R/T790 M) were able to grow without IL-3. Column, mean of independent triplicate experiments; error bars, SD