Figure 1

Lung-associated ALK fusion proteins transform human immortalized lung epithelial cells and are fundamental for the maintenance of the tumor phenotype. (a) NuLi-1 cells were infected with retrovirus expressing different ALK fusion proteins and were collected 96 h after transduction. NPM–ALK and the kinase-dead (EML4–ALK^K589R or TFG–ALK^K231R) fusion proteins were used as controls. Total cell lysates were immunoblotted with the indicated antibodies. (b) NuLi-1 cells transduced with the indicated constructs were plated in soft agar and cultured for 3 weeks. The histograms represent the average numbers of colonies grown in soft agar from the indicated cells and constructs. The number of colonies obtained in TFG–ALK, EML4–ALK or NPM–ALK transduced cells is significantly higher as compared with untransduced cells or to cells transduced with the kinase-dead constructs (*P<0.0005; left panel). Colonies show green fluorescent protein (GFP) positivity (right panel). The number of colonies is normalized to the percentage of infection for every single construct. Cells were plated in triplicate. Results are representative of two different experiments. (c) H2228 TTA A5 and H2228 TTA A5M were cocultured with the untransduced counterpart, respectively, and grown in the presence of 1 μg/ml doxycycline. GFP expression was quantified at indicated time points by fluorescence-activated cell sorting (FACS) analysis and normalized against the initial time point (day 0). Results are representative of two different experiments. (d) H3122, H2228 and H1395 cell lines were treated every 24 h with 300 nM CEP-14083 or CEP-26939. Tetrametylrodamine methyl ester staining followed by FACS analysis was performed after 48 and 72 h of treatment to check the percentage of apoptotic cells. Significance according to the analysis of variance test corresponded to *P<0.05 and **P<0.005. Results are means of duplicates (s.e.m.), and graphics show one representative experiment from two independent assays.