Fig. 4

Reconstitution of TFF1 inhibits IL6-mediated STAT3 activation. a In vitro immunofluorescence assay of AGS pcDNA control cells (Ctrl) and AGS cells stably expressing pcDNA-TFF1. Cells were cultured and treated with or without IL6 (100 ng mL⁻¹). Nuclear localization of STAT3 is shown in green (arrows). DAPI (blue) was used as a nuclear counterstain. Original magnification is at ×400. b Quantification of nuclear STAT3-positive staining in at least 200 cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. c, d Protein-expression levels from the nuclear and cytosolic fractions of AGS (c) and STKM2 (d) cells infected with control or TFF1 adenoviruses (5 MOI). After 48 h, cells were treated or not with IL6 (100 ng mL⁻¹) for 30 min. Similar amounts of protein were applied to SDS-PAGE for immunoblotting with total and p-STAT3 (Y705) antibodies. Anti-NaKATPase antibody was used as a loading control for cytosol fraction, and anti-LAMIN B1 for nuclear fraction. The relative intensity ratio of nuclear p-STAT3/Lamin B1 is calculated and graphed using Image-lab software from BioRad (lower panels). e, f The luciferase reporter assay using a STAT3–Luc reporter plasmid. The results are expressed as mean ± SEM of at least three independent experiments. AGS cells (e) and STKM2 cells (f) infected with control or TFF1 adenoviruses were transfected with STAT3–luciferase reporter and stimulated with IL6 (100 ng mL⁻¹) for 3 h. The results are expressed as mean ± SEM of at least three independent experiments; ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by ANOVA Newman–Keuls multiple comparison test