ALK inhibition-dependent attenuation of MEF-Tet-off-NPM-ALK cell colony formation in soft agar. (A) The MEF-Tet-off-NPM-ALK cells were treated with indicated concentrations of tetracycline for 24 or 48 hours or 17-AAG for 24 hours. The cells were lysed and the lysates were subjected to SDS-PAGE. After transfer, the members were blotted with either phospho-ALK or total ALK antibody. (B-D) The MEF-Tet-off-NPM-ALK cells were treated with indicated concentrations of CEP compounds or 1 μM tetracycline for 24 hours. The cells were diluted with 0.65% agar in culture medium with or without compounds or tetracycline and seeded on 35-mm3 culture dishes at density of 2 × 103/dish. The plates were cultured until individual colonies were formed. The values presented are the average of relative colony numbers from 2 independent assays with standard error. (E) For immunoblotting, the MEF-Tet-Off-NPM-ALK cells were treated with CEP compounds at indicated concentrations for 2 hours and lysed, and the lysates were subjected to SDS-PAGE. After transfer, the membranes were blotted with either phospho-ALK or total ALK antibody.
