Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:52 http://www.jeccr.com/content/29/1/52 Page 4 of 7 Figure 1 Tumor cell killing effect of Ad.hTERT-E1A-TK on NSCLC NCIH460 cells. A. Crystal violet staining of tumor cells after infec- tion with different adenoviral vectors. NSCLC NCIH460 cells were plated into 24-well plates and treated with different doses of adenovi- ral vectors or prodrug or untreated as indicated in figure. 5 days later the plates were stained with crystal violet. B. CCK-8 assay for surviv- ing cells after infection with Ad.hTERT-E1A-TK. NSCLC NCIH460 and A549 cells, and cervical carcinoma Hela cellswere plated into 96- well plates and infected by 10 MOI of Ad.hTERT-E1A-TK with or without 0.5 μg/ml GCV. 5 days later the surviving cells were quantified with CCK-8 assay and normalized by untreated cells. Ad.hTERT-E1A-TK did not cause CPE in primary fibro- blasts but caused CPE in tumor cells which is similar with that in dl309 infected tumor cells (Fig. 2A). In order to confirm that Ad.hTERT-E1A-TK induced tumor specific killing effect was associated with its tumor specific repli- cation, we performed plaque assay to quantify viral prog- eny production. As shown in Fig. 2B, Ad.hTERT-E1A-TK progenies detected in NCIH460 cells were approximately 7000 times more than that detected in primary fibro- blasts. In more detail, about 2 × 107 and 2 × 105 of plaques were detected in supernatant from Ad.hTERT-E1A-TK infected NCIH460 cells and primary fibroblasts at 24 h after infection, whereas on day 5 the plaques were 7 × Figure 2 Selective replication and oncolysis of Ad.hTERT-E1A-TK. A. Comparison of cytopathic effects between NSCLC NCIH460 and primary fibroblasts. NSCLC NCIH460 and primary fibroblasts were plated into 6-well plates and infected with 10 MOI of Ad.hTERT- E1A-TK, dl309 or Ad.GFP. 5 days later cytopathic effects were observed and photographed by light microscopy. B. The virus progeny pro- duction in NCIH460 cells and primary fibroblasts. NCIH460 and pri- mary fibroblasts were infected with 10 MOI of Ad.hTERT-E1A-TK for 4 h then washed once with PBS and then cultured with fresh medium. On 24 h and day 5 post infection, the cells were harvested for plaque assay. The plaques on HEK293 cells were counted and plotted. C. Western blotting analysis of E1A gene expression. NCIH460 and SW1990 Cells were infected with Ad-hTERT-E1A-TK at a MOI of 10. Cell lysates were harvested 48 h later, and immunobloted by anti E1A antibody. HEK293 cells which had been transfected with E1A were used as posi- tive control, and uninfected NCIH460 cells were used as negative con- trol.