Figure 6.

Kaemferol specifically inhibits the phosphorylation of Smad3 at Thr179 that is required for TGF-β1–mediated induction of EMT, cell migration, and MMP2 activation. (A) A549 cells were treated as in Figure 4B. (B) A549 cells treated as in Figure 4A except for the TGF-β1 treatment for 10 minutes. All cells then were subjected to Western blot analysis using specific phosphopeptide antibodies against the phosphorylated Thr179, Ser203, Ser208, and Ser213 in the Smad3 linker region. A549 cells infected with lentivirus carrying expression vector for Myc-tagged Smad3-T179V (Myc-S3^T179V) or its corresponding empty vector (pCAG) were stimulated with 5 ng/ml of TGF-β1 for 36 hours and then subjected to Western blot analysis (C) and immunofluorescence staining (D) using anti–E-cadherin antibody. β-Actin levels were monitored as a loading control for whole extracts. DAPI (blue) was used to stain nuclei. (E) A549 cells were transiently transfected with E-cadherin promoter-reporter construct (E-cadherin-Luc) and Smad3-WT (S3^WT), Smad3-T179V (S3^T179V), or the corresponding empty vector (pcDNA) plasmid DNA and then stimulated with 5 ng/ml of TGF-β1 for 24 hours. All quantitative data are the mean ± SD of three independent experiments. **P < .01. (F) A549 cells infected with lentivirus carrying expression vector for Myc-tagged Smad3 wild type (S3^WT), Smad3-T179V (Myc-S3^T179V), or its corresponding empty vector (pCAG) were stimulated with 5 ng/ml of TGF-β1 for 24 hours and then subjected to ChIP assay using anti-Myc antibody. The purified DNA was analyzed as in Figure 3D. (G) A549 cells were transiently co-transfected with Snail promoter-reporter (Snail-Luc) and Smad3-WT (S3^WT), Smad3-T179V (S3^T179V), or the corresponding empty vector (pcDNA) plasmid DNA and then stimulated with 5 ng/ml of TGF-β1 for 24 hours. All quantitative data are the mean ± SD of three independent experiments. **P < .01. (H) A549 cells (1 × 10⁴) that were infected as in Figure 5B were seeded on 8-mm porous Transwell chambers and then stimulated with 5 ng/ml of TGF-β1 for 36 hours. Transmigrating cells were stained and counted as in Figure 2B. Quantitative data are shown as the mean ± SD of three independent experiments. **P < .01. (I) A549 cells were transiently transfected with MMP2 promoter-reporter construct (MMP2-Luc) and Smad3-WT (S3^WT), Smad3-T179V (S3^T179V), or the corresponding empty vector (pcDNA) plasmid DNA and then stimulated with 5 ng/ml of TGF-β1 for 24 hours. All quantitative data are the mean ± SD of three independent experiments. **P < .01.