Figure 7

Sp1 positively regulates E-cadherin expression and attenuates the translocation of β-catenin into the cell nucleus. (a) Whole-cell extracts of CL1-0 and CL1-5 cells were collected for western blotting with antibodies against E-cadherin, β-catenin and β-actin. (b) Binding of Sp1 to the promoter of E-cadherin was determined by chromatin immunoprecipitation (CHIP) assay. (c) Effect of Sp1 knockdown on the protein level of E-cadherin in lung adenocarcinoma cells with low invasiveness. After transfection of scrambled or Sp1 small hairpin RNA (shRNA) for 48 h, whole-cell extracts of CL1-0 and A549 cells were collected for western blotting with antibodies against E-cadherin and β-actin. Quantitated results are shown in the lower panel. Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05, **P<0.01). (d) Effect of Sp1 on the protein levels of E-cadherin and β-catenin in lung adenocarcinoma cells with high invasiveness. After infection with GFP or increasing dosage (1–20 m.o.i.) of GFP-Sp1 adenovirus for 48 h, whole-cell extracts of CL1-5 cells were collected for western blotting using antibodies against E-cadherin, β-catenin and β-actin. pSp1 represents phosphorylated Sp1. Quantitated results are shown in the lower panel. Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05, **P<0.01). (e) Effect of Sp1 on the translocation of β-catenin into nucleus. (Left panel) After infection with GFP or increasing doses of GFP-Sp1 adenovirus for 48 h, cytosolic and nuclear fractions were isolated for western blotting with antibodies against β-catenin, tubulin and histone H3. (Right panel) Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05, **P<0.01). (f) Immunofluorescent staining of nuclear GFP-Sp1 and β-catenin in CL1-5 cells. After infection of cells with adenovirus-expressing GFP-Sp1 for 48 h, cells on a coverslip were stained with an anti-β-catenin antibody and DAPI. Stained cells were photographed under a fluorescence microscope at × 1000 magnification. (g) Effect of Sp1 on the expression of T-cell factor-4 (Tcf4), a target gene of β-catenin. After infection of cells with adenovirus expressing GFP-Sp1 for 48 h, RNA was prepared for reverse transcriptase–PCR (RT–PCR). (h) The proposed model showing that Sp1 blocked β-catenin translocation into the cell nucleus through inducing E-cadherin, which was shown to anchor β-catenin near plasma membrane.