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