Fig. 3
EML4-ALK promotes anchorage-independent growth in hTERT-transduced normal human fibroblasts. a hTERT-CRL-2097 were transduced with the EML4-ALK lentiviral vector (for the constitutive expression in the absence of a Tet repressor) or the control vector (Vec). Western blot analysis was performed in the cells at indicated PDL after transduction, as in Fig. 1a, with H3122 as a positive control. b Cell proliferation curves of hTERT-CRL-2097 with the expression of EML4-ALK or the control vector. Cumulative PDL were calculated and plotted to days after transduction, as above. c No accumulation of DNA damage by EML4-ALK in hTERT-transduced fibroblasts. The hTERT-CRL-2097 cells expressing EML4-ALK (PDL 12), with the control vector (PDL 10) and before transduction were examined for γ-H2AX foci as in Fig. 1e. The CRL-2097/TR fibroblasts at senescence (Vector at PDL 12 in Fig. 1b and e) were again examined for comparison and shown in parallel. The data quantification and analysis were as in Fig. 1e. * P < 0.05; *** P < 0.001. d Anchorage-independent growth of hTERT-CRL-2097 cells expressing EML4-ALK, with and without 25 nM Crizotinib. Those cells with the control vector were plated in soft-agar medium and examined for colony formation at day 21. Numbers of colonies per 1 × 103 cells plated (means ± s.d. from biological triplicate) are shown with representative images without or with a colony (arrow). Scale bars, 50 μm. ** P < 0.01. e Summary of karyotype and the status of the TP53 gene in EML4-ALK-expressing hTERT-CRL-2097 fibroblasts and their derived cell clones isolated and established from soft-agar colonies (#1 to #6). n.d., not determined