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. 2019 Dec 9;20(24):6215. doi: 10.3390/ijms20246215

Figure 2.

Figure 2

Depleted expression of TFF3 decreases oncogenic behaviour in SW620 cells. SW620 cells were transiently transfected with TFF3 siRNA (designated SW620-siTFF3) or scrambled siRNA (SW620-siSC). (A) Detection of TFF3 expression by qPCR and Western blot analysis. β-ACTIN was used as input control. (B) Total cell count. Cells were seeded in six-well plates in triplicate at 10 × 104 cells/well on day 0. Cell numbers were counted at the indicated time points. (C) Cell cycle progression of cells cultured in 2% FBS medium was determined using 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) Foci formation. Cells were seeded in six-well plates and cultured for 10 days prior to fixation and crystal violet staining. (G) 3D Matrigel growth. Cells were cultured in 5% FBS medium containing 4% Matrigel. Cell viability was determined by AlamarBlue assay after eight days. Fold change of cell viability relative to –Vec cells is shown in the histogram. Representative microscopic images of viable colonies formed by the respective cells in 3D Matrigel and stained by CellTrace Calcein Green AM are shown. Scale bar: 200 μm. (H) Cell migration assay. Cells that migrated across the Transwell membrane after 12h were stained with Hoechst 33342 and counted under the fluorescence microscope. Fold change of migrated cells relative to –Vec cells is shown in the histogram. (I) Cell invasion assay. 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 –Vec cells is shown in the histogram. Data are expressed as mean ±SD. **, p < 0.01; ***, p < 0.001.