Figure 2. Anti-tumor effects of 4.1N on the proliferation, migration and adhesion in H1299 and 95C cells in vitro.

Both 95C /H1299 cells were transiently transfected with the same amount of human-4.1N shRNA/pEGFP-4.1N or with the negative control mouse-4.1N shRNA/null pEGFP vector. (A) Cell proliferation was measured by MTT assay for 24 h, 48 h and 72 h after cell plating. The degree of proliferation was reflected by the optical density (OD) value at 570 nm; the larger OD value indicated more active cell proliferation. The data are presented as the mean ± standard deviation (SD) from three independent experiments. Top panel: the effectiveness of the plasmids on 4.1N expression was evaluated by Western blotting analysis. (B) More than 90% of the confluent monolayer of transfected cells was scratched and imaged by light microscopy at three time points 0, 12, and 24 h. The degree of motility was shown as percent of wound closure as compare with the 0 h time point. (C) The migration of transfected H1299 and 95C cells was measured by using transwell chambers. Representative images of cells penetrating the chamber membrane were shown. The results are expressed as the average number of cells in five random microscopic fields ± SD of three independent experiments. (D) Transfected cells were seeded into 96-well plates that were pre-coated with fibronectin and incubated for 1 h at 37°C. Nonadherent cells were washed away, while the adherent cells were stained with crystal violet; the absorbance of the released crystal violet after extraction with SDS was quantified by spectrophotometry at 570 nm. The results are the mean ± SD from three independent experiments. *indicates p < 0.05 versus control based on the Student's t-test.