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