ALK siRNA reverts NPM-ALK–mediated transformation of MEF cells in vitro and in vivo. (A) Suppression of NPM-ALK expression leads to down-regulation of known downstream targets of ALK. Lysates from NPM-ALK Tet-Off MEFs infected with pSRG-A5 virus (semiconfluent cells in absence of doxycycline) before (40% EGFP⁺) or after (> 90% EGFP⁺) puromycin selection were immunoblotted with the indicated antibodies. (B) NPM-ALK MEF cells lose their transformed phenotype in absence of NPM-ALK expression. Cell cultures in absence of doxycyline are rescued from the ALK-mediated transformed phenotype by the expression of ALK-A5 shRNA (contrast phase microscopy, left panels). Control and pSRG-A5–infected NPM-ALK Tet-Off MEF cells were stained with anti-ALK antibodies, followed by biotin-conjugated horse antimouse antibody and streptavidin-Cy3. Cells were counterstained with Hoechst 33258 and visualized with a Leica fluorescence microscope using a 63× objective (right panels). (C) pSRG-A5 expression prevents NPM-ALK MEF cell growth in immunocompromised mice. NPM-ALK Tet-Off MEF cells transduced with pSRG-A5 or control pSRG were first selected with puromycin (> 90% EGFP⁺), inoculated (10⁶ cells/mouse) subcutaneously into athymic Nu/Nu mice recipients (3 mice for each construct). • indicates pSRG; ▪, pSRG-A5. Tumor growth was monitored over time. These findings are representative of 2 experiments. Error bars indicate SD.