NPM-ALK inhibits p53 activity and it induces a senescent checkpoint in primary fibroblasts. (A-F) WT MEFs were infected with control (Ctrl) and NPM-ALK (NA) or RASV12 expressing retroviruses. (A) Cells were analyzed (5 days after infection) by immunofluorescence (magnification ×600) and Western blot (B) with an anti-γH2AX antibody (representative micrographs and percentage of labeled cells are shown). (C) Growth curve of infected cells plated after infection and counted daily (cell numbers are expressed relative to day 1; *P < .05 relative to control cells infected with an empty vector). (D) β-galactosidase detection (representative micrographs [magnification ×600] and percentage of positive cells) and anti-BrdU staining (percentage of positive cells relative to control cells) performed 6 and 4 days after infection, respectively. (E) Western blotting of p53, ARF, p16INK4a, NPM-ALK, and tubulin expression, 4 days after infection. (F) Western blotting of pRb, NPM-ALK, cyclin A, and tubulin expression, 3 days after infection. (G) Luciferase assay in WT MEFs transduced with the pGL13 p53-reporter, the CMV-βGal vector, and increasing amounts of vectors expressing NPM-ALK or the K210R kinase-dead NPM-ALK mutant (or a control empty vector). Data are from 3 independent experiments and normalized to βGal. *P < .05 relative to control cells infected with an empty vector. (H) Same Luciferase assay as in panel G comparing a single dose (100 ng) of NPM-ALK– and RASV12-expressing vectors.