Fig 2: Establishment of TFF-1-overexpressing cell lines. SBC-5 and LC-AI cells were transfected with a vector containing the cDNA encoding TFF-1/FLAG (pBA-TFF1) or empty vector (pBA-neo). (A) Expression of this fusion protein was evaluated by immunoblotting analysis with anti-TFF-1 antibody. Expression was not observed in parental SBC-5 or LC-AI cells (BC), but a single band of around 15kDa was detected after transfection of pBA-TFF1 (G-TFF1) in 4 clones, but not observed in cells transfected with pBA-neo (G-neo). Expression was normalized to the ß-actin loading control. “Marker” indicates molecular weight marker. (B and C) Images of immunofluorescent staining in parental cells (BC), pBA-neo (G-neo) or pBA-TFF1-transfected cells (G-TFF1) of SBC-5 (B), LC-AI (C) cells are shown. Cells were stained with anti-TFF-1 antibody and with 4',6-diamidino-2-phenylindole (DAPI). Scale bar indicates 100µm. (D) Schematic presentation of TFF-1 mRNA expression after transfection with pBA-neo or pBA-TFF1 (3 clones; CL) expressed relative to values obtained for H1975. Square and bar indicate mean values and standard deviations, respectively, obtained by qRT-PCR analysis from three replicated experiments with three replicate samples. Results were statistically analyzed and p-values were obtained by Fisher’s PLSD test.

Fig 3: Constitutive overexpression of TFF-1 suppresses cell proliferation and enhances cell death. (A) Growth curves of parental, pBA-neo, or pBA-TFF1-transfected SBC-5 and LC-AI cells during 7 days observation. (B) Increase in dead SBC-5 and LC-AI cells in these transfection groups. (C) Schematic presentation of living (blue) and dead (red) cell fractions of cultured cells counted by hemocytometer. (D) Results of viability assay in the 3 transfection groups measured by colorimetric Viability/Cytotoxicity Multiplex Assay Kit. (E) Results of dead cell assay measured by the same kit. (A–E) Mean value ± standard deviation (bar), obtained from three replicated experiments, were indicated. Results were statistically analyzed by one-way ANOVA and p-values were obtained by Mann–Whitney U-test.

Fig 4: The type of cell death analyzed by immunofluorescent staining for active caspase 3/7 (A and B). Representative fields of parental, pBA-neo-transfected and pBA-TFF1-transfected cells stained for active caspase 3/7 (green) and DAPI (blue) after 7 days of culture. The value indicates the ratio of caspase-active to DAPI-stained cells obtained from three replicated experiments. Overexpression of TFF-1 resulted in a marked increase in the fraction of caspase-active cells, while not induced by transfection of pBA-neo. Scale bar indicates 100µm.

Fig 5: siRNA against TFF-1 reversed the suppression of cell proliferation and cell death caused by TFF-1. (A) Suppression of TFF-1 expression by transfection of TFF-1-specific and non-targeting siRNA into TFF1-transfected cells in the absence or presence of 2% serum in the media. Square and bar indicate mean values and standard deviations, respectively, obtained from three replicated experiments with three replicate samples analyzed by ELISA. Results were statistically analyzed and p-values were obtained by Fisher’s PLSD test. (B) Growth curves of parental, pBA-neo, or pBA-TFF1-transfected SBC-5 and LC-AI cells with or without transfection by TFF-1 siRNA or non-targeting siRNA evaluated by viability assay. (C) Dead cell fractions evaluated by cytotoxicity assay in the same SBC-5 and LC-AI-derived cell lines. (B and C) Mean value ± standard deviation (bar), obtained from three replicated experiments, was indicated.