Lentiviral ALK shRNA abrogates NPM-ALK expression and signaling. (A) Lentivirus infection with ALK-A5 leads to an efficient down-regulation of NPM-ALK protein expression. TS cells were transduced with ALK sh-RNA lentivirus (300 μL) and then harvested to determine the NPM-ALK protein expression over time. Immunoblotting with anti-GFP antibodies was used to check the expression of the report gene within the lentiviral cassette. (B). Loss of NPM-ALK is specifically observed in cells infected with ALK-A5. TS cells were transduced with ALK-A5 or TM2 viruses, cultured for 96 hours, and then stained with anti-ALK antibody. Immune complexes were visualized using biotin-conjugated rabbit antimouse antibody followed by streptavidin-Cy3 (right panels). Nuclei were identified using Hoechst 33258 (left panels). EGFP expression is shown in middle panels. Objective magnification, 40×. (C) TS cells transduced with ALK-A5 show phospho-Stat3, phospho-AKT, and Jun B down-modulation. EV indicates empty vector.