Effects of TIG1A and TIG1B expression on β-catenin/TCF- and CREB-mediated activities and β-catenin subcellular localization on TIG1A- and TIG1B-expressing HCT116 cells. ( A) β-catenin/TCF (top panel)- and CREB (bottom panel)-mediated activities were analyzed using luciferase reporters. Indicated stable cells were transfected with the reporter plasmid and then incubated with 5 nM MFP for 24 h, serum starved for 16 h, and then treated with or without PGE2 (10 nM) for 24 h. MFP was present during serum starvation and PGE2 treatment in all similar experiments. Cellular lysates were analyzed for transactivation activity of the TOPFLASH or CREB luciferase reporters. Results were expressed as means ± SD from triplicate samples after normalization to protein concentration and then to the results obtained from TIG1A-inducible cells without PGE2 treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Effects of TIG1 isoforms on β-catenin subcellular distribution were determined by subcellular fractionation and Western blot analysis. Indicated stable cells were incubated with 5 nM MFP for 24 h, serum starved for 16 h, and incubated with or without 10 nM PGE2 for 30 min. After subcellular fractionation, β-catenin, TIG1A, TIG1B, lamin B1, and GAPDH levels were determined by Western blot analysis. Normalization of nuclear (N) and cytosolic (C) β-catenin levels was described in the Materials and Methods section, and the respective cells without PGE2-treatment were designated as 1.0. (C) Effects of TIG1 isoform expression on β-catenin subcellular distribution was determined by immunofluorescent analysis. Stable cells were incubated with MFP, serum starved, and incubated with PGE2 for 30 min as described above. β-catenin localization (green) and nuclei (blue) were analyzed using a laser scanning confocal microscope. Bars, 10 μm. Arrows indicate nuclear β-catenin.