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Fig. 4. Caveolin-1 suppressed ß-catenin-Tcf/Lef-dependent transcription of survivin in HEK293T cells. HEK293T cells were transfected with the plasmids pLacIOP or pLacIOP-caveolin-1 and subjected to further analysis after 24 hours. (A) Cellular localization of caveolin-1 (upper panels) and ß-catenin (middle panels) was detected by confocal microscopy; merged images (bottom panels). Bar, 10 µm. Note that co-localization was predominantly detected at the plasma membrane cell-cell contact sites (white arrows). (B) Ectopically expressed caveolin-1 was immunoprecipitated from HEK293T cells and then caveolin-1 and ß-catenin were detected by western blotting. A result representative of two independent experiments is shown. (C) HEK293T cells were co-transfected with the plasmids pLacIOP (vector) or pLacIOP-caveolin-1 (cav-1) and pTOP-FLASH (black bars) or pFOP-FLASH (white bars) in the presence or absence of 20 mM LiCl. After 24 hours, cell extracts were prepared and utilized for Tcf/Lef reporter assays (bar graph) and western blot analysis. Bar graph: luciferase activity (normalized to ß-galactosidase) as a percentage of the values detected in vector-transfected cells (pLacIOP, defined as 100%). Data shown are mean ± s.e.m. from three independent experiments; *comparison with pLacIOP control, P<0.01. Western blot: determination of survivin and actin levels. From left to right: cells transfected with pLacIOP (vector) or pLacIOP-caveolin-1 (cav-1) in the absence (-) or presence (+) of LiCl (20 mM). (D) HEK293T cells were co-transfected with the plasmids pLacIOP (black bars) or pLacIOP-caveolin-1 (hatched bars) and the reporter constructs pLuc-1710, pLuc-420, pLuc-420-2M or pLuc-420-3M as indicated. After 24 hours, cell extracts were prepared and used to measure reporter activity. Data shown are mean ± s.e.m. from three independent experiments; #comparison with pLuc-1710 in the presence of pLacIOP, P<0.001; 
comparison with pLuc-420 in the presence of pLacIOP, P<0.01.
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