The adaptor p130Cas is required for NPM-ALK–mediated transformation and actin filaments organization. NIH3T3 MEFs and Cas^-/- MEFs were infected with Pallino NPM-ALK or Pallino NPM-ALK^K210R retroviruses and sorted to obtain greater than 95% GFP-positive cells. (A-B) The expression of NPM-ALK in NIH3T3 MEFs led to spindle-shape morphology and to the outgrowth of cellular processes. NPM-ALK^K210R was used as a control. (C-D) Cas^-/- cells infected with Pallino NPM-ALK did not show evident morphologic differences in comparison with the control NPM-ALK^K210R. Phase-contrast images acquired with a 40×/0.55 objective lens. (E-H) Cas^-/- fibroblasts expressing NPM-ALK retain the organization of the actin cytoskeleton. Compared with NPM-ALK NIH3T3 MEFs that undergo an evident loss of the actin structure (F), NPM-ALK Cas^-/- fibroblasts show a phenotype resembling Cas^-/- infected with the control NPM-ALK^K210R (G-H). Cells were incubated with PE-conjugated phalloidin to stain the actin filaments (red). Images were taken with the Leica confocal microscope equipped with a 63×/1.32 oil immersion objective lens. (I) Protein expression levels were analyzes by WB as indicated. (J) Cell-cycle analysis was performed by DNA content evaluation on cells in logarithmic growth phase. (K) NIH3T3 and Cas^-/- MEFs, infected as indicated, were plated in soft agar and cultured for 3 weeks. The histograms represent the average numbers of colonies from the indicated cells and constructs. Data are from 1 of 3 independent experiments, each including triplicates for experimental point.