Fig. 1. Forced expression of TFF3 promotes oncogenicity of H1299 and H1975 cells.

H1299 or H1975 cells were stably transfected with an expression vector containing the TFF3 cDNA (designated H1299-TFF3/H1975–TFF3) or pIRESneo3 vector alone (H1299-Vec/H1975-Vec). a Detection of TFF3 expression by RT-PCR and western blot. cm = conditioned media. β-ACTIN was used as input control. b The total cell count in RPMI-1640 media supplemented with 10% or 0.2% FBS. Cell numbers were counted at the indicated time points. c Cell-cycle progression of cells cultured in 10% FBS medium was determined using propidium iodide (PI) staining followed by FACs analysis. The percentages of cells in each cell-cycle phase are plotted. d Annexin-V/PI apoptotic cell death was determined after 24 h serum deprivation. The percentages of early apoptotic (Annexin-V-positive/PI-negative) and late apoptotic (Annexin-V-positive/PI-positive) cells are plotted. e Caspase 3/7 activities in the cells were determined after 24 h serum deprivation. f Soft agar colony formation. Cells were seeded in 0.35% agarose in full medium on a base layer of 0.5% agarose. Complete culture medium was added and replaced every 3 days. Colonies formed after 14 days were counted and fold change in colony numbers relative to the respective –Vec cells are shown in the histogram. g Foci formation. Cells were seeded in six-well plates, and cultured for 2 weeks prior to fixation and crystal violet staining. h 3D Matrigel growth. Cells were cultured in 5% FBS medium containing 4% Matrigel. Cell viability was determined by AlamarBlue assay after 9 days. Fold change of cell viability relative to the respective –Vec cells is shown in the histogram. i Spheroid-formation assay. Cells were seeded in ultralow attachment plates in spheroid growth media. After 10 days, spheroid growth was measured by AlamarBlue assay, and the fold change of cell viability relative to the respective –Vec cells is shown in the histogram. j Cell-migration assay. The number of cells that migrated across the transwell membrane after 12 h were stained with Hoechst 33342 and counted under the fluorescence microscope. Fold change of migrated cells relative to the respective –Vec cells is shown in the histogram. k Cell invasion assay. The number of cells that invaded across the 10% Matrigel-coated transwell membrane after 24 h were stained with Hoechst 33342 and counted under the fluorescence microscope. Fold change of invaded cells relative to the respective –Vec cells is shown in the histogram. The data are expressed as mean ± S.E.M. *p < 0.05; **p < 0.01