Morphologic changes induced by NPM-ALK. (A-B) MEFs Tet-Off show spindle-transformed cell shape when NPM-ALK is expressed. MEFs Tet-Off were stably transfected with the inducible vector NPM-ALK pBIEGFP and forced to express NPM-ALK when grown in medium deprived of tetracycline for 48 hours. (C-D) The 293 T-Rex Tet-On cells undergo changes in adherence when NPM-ALK is induced by adding tetracycline to the culture medium for 24 hours. Contrast-phase images; 40×/0.55 objective lens. (E-F) NIH3T3 fibroblasts were infected with Pallino NPM-ALK retrovirus and then incubated with monoclonal antipaxillin primary antibody followed by FITC-conjugated secondary antibody (green) and with PE-conjugated phalloidin to stain the actin filaments (red). Noninfected NIH3T3 cells (E) show a spread morphology, with clearly detectable actin filaments ending in the focal contacts (red arrows) and with organized paxillin clusters (green arrows), whereas NIH3T3 NPM-ALK cells (F) show only few actin filaments (red arrow) and rare paxillin clusters (green arrows). Images were taken with the Leica confocal microscope using a 63×/1.32 objective lens. Right panels show NPM-ALK expression by WB in the corresponding cells. Samples were blotted with anti-ALK monoclonal antibody. (G) CEM cells were infected with retroviruses expressing NPM-ALK or the kinase-dead mutant NPM-ALK^K210R as control and then sorted for GFP expression. The histograms represent the numbers of cells migrated in response to SDF-1α in a transwell assay. The histograms summarize the results of 3 independent experiments using triplicate wells for experimental point. The right panels show NPM-ALK expression and phosphorylation by WB in the corresponding cells. Samples were blotted with anti-ALK and anti-PY20 monoclonal antibodies. (H) NPM-ALK silencing by ALK-shRNA decreases the migration rate of TS cells. TS cells were cotransduced with pLVTH ALK-shRNA and pLV-tTRKRAB vectors to obtain a doxycycline-dependent inducible NPM-ALK silencing. Cells were grown in the presence of doxycycline (1 μg/mL) for 72 hours. The histograms represent the numbers of cells migrated in response to SDF-1α in a transwell assay. The histograms are from 3 independent experiments using triplicate wells for experimental point. The right panels show NPM-ALK expression by WB in the corresponding cells. Samples were blotted with anti-ALK monoclonal antibody. ^*Statistically significant as analyzed by Student t test.