Miyanaga A , Matsumoto M , Beck JA , Horikawa I , Oike T , Okayama H , Tanaka H , Burkett SS , Robles AI , Khan M , Lissa D , Seike M , Gemma A , Mano H , Harris CC .

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	Fig. 1. EML4-ALK induces early onset of cellular senescence in mortal normal human fibroblasts. CRL-2097/TR were transduced with an inducible lentiviral vector of EML4-ALK or its control vector (Vec). Shown PDL is after transduction. a Western blot analysis confirming the inducible expression of EML4-ALK. The EML4-ALK- or Vec-transduced cells with (+) or without (−) doxycycline induction (Dox) were examined at indicated PDL for protein expression levels of total EML4-ALK and phosphorylated EML4-ALK. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was a loading control. H3122 (EML4-ALK expressing cells). Full-length blots are presented in Supplementary Figure. b Replicative lifespan of transduced CRL-2097/TR fibroblasts. Cumulative PDL after transduction were plotted to days after transduction. According to ATCC’s PDL counting, CRL-2097/TR at the initiation of this experiment corresponded to PDL 43 in total. The proliferation arrest of Vector or Dox (−) control cells at PDL 11 or 12 after transduction, corresponding to PDL 54 or 55 in total, is consistent with the information from ATCC that this strain senesces at PDL 56. c SA-β-gal staining of CRL-2097/TR fibroblasts with (Dox+) and without (Dox-) induced expression of EML4-ALK. Representative images in proliferative phase (both at PDL 4) and growth arrested phase (Dox + at PDL 6 and Dox- at PDL 11) are shown. Percentages of SA-β-gal positive cells (means ± s.d.) were from biological triplicate, in each of which more than 300 cells were observed. Scale bars, 20 μm. d Western blot analysis of p16INK4A and p21WAF1. The EML4-ALK-transduced CRL-2097/TR fibroblasts with (+) and without (−) Dox induction at indicated PDL, in parallel to the cells before transduction (Pre), and those retrovirally transduced with H-RasV12 and its control vector pBabe (Supplementary Fig. S1A-B) were examined. Quantitative expression levels of p16INK4A and p21WAF1 (normalized with GAPDH) are shown in relative to the levels in the cells before transduction (Pre). e Early and accelerated accumulation of DNA damage in cells expressing EML4-ALK. Senescent CRL-2097/TR with control vector (PDL 12) and those with Dox-induced EML4-ALK expression (PDL 6) were examined for γ-H2AX foci. The data (signal intensity per cell) are mean ± s.d. from triplicates and shown relative to the vector control. f Representative images of γ-H2AX immunofluorescence staining in the cells shown in (e). The lower panels were merged with DAPI. CRL-2097/TR with control vector (PDL 7) is also shown as comparison. Note that the level of γ-H2AX foci in natural replicative senescence (Vector, PDL 12) is consistent with that previously reported [25]. Scale bars, 20 μm
	Fig. 2. The kinase activity of EML4-ALK is required for early induction of cellular senescence. a Western blot analysis showing a decrease in EML4-ALK autophosphorylation by an ALK TKI Crizotinib. CRL-2097/TR fibroblasts with Dox-induced expression of EML4-ALK were maintained in culture in the absence (−) or presence (+) of 25 nM Crizotinib and examined for levels of total EML4-ALK (top) and phosphorylated EML4-ALK (middle). Dox was added at day 0 and Crizotinib was added at day 5 and both remained included throughout the experiment with medium change every 48 h. The cells were harvested at day 40 (at PDL 6 or 10) for western blot. b Abrogation of early senescence by Crizotinib. The Dox-treated cells (Dox+) with and without Crizotinib (+ and -) shown in (a), along with the untreated cells (Dox-), were examined for cumulative PDL after transduction as in Fig. 1b. c Western blot analysis of MRC-5 fibroblasts that express wild-type EML4-ALK and the kinase-dead mutant (K589M). MRC-5, a second strain of mortal normal human fibroblasts, were transduced with the lentiviral vector of EML4-ALK (the same vector as used above, which drives constitutive expression in the absence of a Tet repressor) and its K589M mutant derivative. Western blot analysis was performed as in (a). d Dependence of early senescence on the kinase activity of EML4-ALK. The transduced MRC-5 fibroblasts shown in (c), along with the vector control-transduced cells, were examined for cumulative PDL after transduction, as in (b) and Fig. 1b. Note that this experiment used late-passage MRC-5 with fewer PDL remaining until natural replicative senescence, compared with CRL-2097/TR used in the above experiments. e Representative images of SA-β-gal staining with quantitative data of positive cells (mean ± s.d., as in Fig. 1c). The cells shown in (d) were examined at day 22. Scale bars, 20 μm
	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
	Fig. 4. STAT3 is phosphorylated by EML4-ALK in both mortal and hTERT-transduced normal human fibroblasts. The same set of CRL-2097/TR-derived cells as in Fig. 1a (a) and the same set of hTERT-CRL-2097-derived cells as in Fig. 3a (b) were examined in western blot analysis for levels of total and phosphorylated STAT3, total and phosphorylated Akt, total and phosphorylated Erk1/2, and total and phosphorylated Src. Full-length blots/gels are presented in Supplementary Figure
	Fig. 5. EML4-ALK promotes anchorage-independent growth in hTERT-immortalized normal human bronchial epithelial cells. a HBET1, an hTERT-immortalized normal human bronchial epithelial cell line, was transduced with the EML4-ALK lentiviral vector or the control vector as in Fig. 3. The cells at 28 days after transduction were examined in western blot analysis as in Figs. 3a and 4. Full-length blots/gels are presented in Supplementary Figure. b Cell proliferation curves of HBET1 expressing EML4-ALK or with the control vector. Cumulative PDL were calculated and plotted to days after transduction, as above. c Anchorage-independent growth of HBET1 cells expressing EML4-ALK. The HBET1 cells with EML4-ALK or the control vector were examined for anchorage-independent growth, as in Fig. 3d and S5C. Data analysis and presentation are also as in Fig. 3d and S5C. Arrows in a representative image indicate colonies that formed in soft-agar medium. Scale bars, 50 μm. ** P < 0.01. d Summary of tumorigenicity assay in NOD.SCID/Ncr mice. Cells were subcutaneously injected into each flank (5 × 106 per flank) of mice at 6–10 weeks of age, followed by observation until 8 weeks after injection. In each mouse, one flank had EML4-ALK-expressing cells or soft-agar clones, and the other flank had cells with the control vector. Whereas NIH/3 T3 cells expressing EML4-ALK (2 × 106 per flank, at both flanks of two mice) produced tumors necessitating euthanasia, no progressively growing tumors formed from any of hTERT-CRL-2097- and HBET1-derived cells

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