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. 2019 Nov 4;8(11):65. doi: 10.1038/s41389-019-0173-8

Fig. 2. Depleted expression of TFF3 decreases oncogenicity of H1299 and H1975 cells.

Fig. 2

H1299 or H1975 cells were transiently transfected with TFF3 siRNA (designated H1299-siTFF3/H1975-siTFF3) or scrambled siRNA alone (H1299-siSC/H1975-siSC). a Detection of TFF3 expression with western blot analysis. β-ACTIN was used as an input control. b The total cell count in RPMI-1640 media supplemented with 10% FBS. Cell numbers were counted at the indicated time points. c Cell-cycle progression of cells cultured in 10% FBS medium was determined using PI staining followed by FACs analysis. The percentages of cells in each cell-cycle phase are plotted. d Caspase 3/7 activities in the cells were determined after 24 h serum deprivation. e 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. Representative microscopic images of colonies formed by the respective cells in soft agar are shown. Scale bar: 200 μm. f 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. Representative microscopic images of colonies formed by the respective cells in 3D Matrigel are shown. Scale bar: 100 μm. g Spheroid-formation assay. Cells were seeded in ultralow attachment plates in spheroid growth media. After 10 days, spheroid growth was measured by AlamarBlue, and the fold change of cell viability relative to the respective –Vec cells is shown in the histogram. Representative microscopic images of spheroids formed by the respective cells are shown. Scale bar: 20 μm. h Cell-migration. 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. i 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