Fig 1: Loss of TFF1 promotes STAT3 nuclear localization in the TFF1-KO mice gastric tissues. a Representative immunohistochemistry images in the upper panels show nuclear p-STAT3 (Y705) in hyperplasia, dysplasia, and cancer from the antropyloric region of glandular stomach in TFF1-knockout (TFF1-KO), but not in TFF1-wild-type (TFF1-WT) normal gastric mucosa. The lower panels demonstrate the expression of TFF1, original magnification (×200) and insets (×400). b Immunohistochemistry staining of p-STAT3 of gastric mouse tissues from TFF1-WT and TFF1-KO mice at the age of 2, 6, and 10 months detected p-STAT3 in the nucleus at all ages in TFF1-KO mice. p-STAT3 staining was absent in tissues from matched ages in TFF1-WT mice with normal glands. The TFF1-KO mice showed dysplastic lesions at 6 and 10 months of age. Original magnification (×400) and insets (×600)
Fig 2: Loss of TFF1 promotes mRNA expression of STAT3 target genes in gastric tumourigenesis. a Quantitative real-time PCR analysis demonstrated upregulation of mRNA expression of STAT3 target genes (Vegf, c-Myc, Birc5, and Il17A) in gastric tissues from the TFF1-KO mice (n = 36) as compared with normal gastric tissues from TFF1-WT mice (n = 15). b Expression levels of STAT3 target genes in nondysplastic and dysplastic gastric mucosa from TFF1-KO, as compared to normal gastric mucosa from TFF1-WT. c Expression levels of STAT3 target genes in TFF1-KO gastric mucosa at 2–4 and >6 months of age, as compared to age-matched TFF1-WT gastric mucosa. Horizontal bars indicate the mean values. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student’s t test (for two groups) and ANOVA Newman–Keuls multiple comparison test (for multiple groups)
Fig 3: TFF1 negatively regulates IL6-induced STAT3 activation through GP130-IL6Rα axis. a Western blot analysis using AGS cell lines infected with control or TFF1 adenovirus. After stimulation with IL6 (100 ng mL−1) for 30 min, TFF1 expressing cells showed a significant decrease of p-STAT3 (Y705), p-GP130 (S782), and p-JAK2 (Y1007/Y1008) protein levels as compared to control cells. b The relative intensity ratio of p-JAK2/β-Actin, p-GP130/β-Actin, and p-STAT3/β-Actin were calculated by Image-lab software from BioRad. The results are expressed as mean ± SEM of at least three independent experiments. ***P < 0.001 by two-tailed Student’s t test. c Immunoprecipitation and Western blot analysis following IL6Rα pulldown using AGS cells infected with TFF1 or control adenoviruses (5 MOI), with or without treatment with IL6 (100 ng mL−1) for 30 min. The first lane exhibits AGS control following immunoprecipitation with mouse IgG control antibody. All immunoprecipitations and their corresponding input samples were subjected to immunoblotting with rabbit polyclonal antibody against GP130 and IL6Rα. The expression of TFF1 and equal amounts of protein loading were confirmed in the input samples. d Punctate proximity ligation assay (SOURCE) was performed in accordance with supplier’s instructions in AGS cells infected with control or TFF1 adenovirus. The presence of red signals indicates positive ligation, indicative of interaction. Using IL6Rα and GP130 antibodies, the results indicated the presence of IL6Rα–GP130 interaction (red signals), following stimulation with IL6 in control cells (Ctrl). This interaction was not detected in AGS cells expressing TFF1 (upper left two panels). The lower panel displays immunofluorescence following the use of TFF1 and IL6Rα antibodies and demonstrates an interaction between TFF1 and IL6Rα (red signals), (lower right two panels). As a negative control for PLA background reaction, control cells were stimulated with IL6 and probed with a single antibody for GP130 (right single panel). Maximum intensity projection is presented in the upper and right side of each image
Fig 4: TFF1 suppresses expression of STAT3 target genes in vitro. a–c Quantitative real-time PCR analysis demonstrated expression levels of several STAT3 target genes (VEGF, C-MYC, CXCL10, and IL17A) in vitro. a AGS cell lines were infected with either TFF1 or control adenoviruses (5 MOI) for 48 h and then treated with or without IL6 (100 ng mL-1) for 2 h. b AGS cells were treated with or without TFF1 recombinant protein (400 ng mL-1) for 24 h and either stimulated or not with IL6 for 2 h. c AGS cells were treated with conditioned media from AGS-pcDNA or AGS-TFF1 stable cell lines for 24 h. The results are expressed as mean ± SEM of at least 3 independent experiments; *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student’s t test (for two groups) and ANOVA Newman–Keuls multiple comparison test (for multiple groups)
Fig 5: 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
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