Figure 4

Effects of TIG1 and GRK5 siRNAs on TIG1-mediated suppression of reporter activities and nuclear β-catenin accumulation. (A) Western blot analysis of HCT116 cell lysates transiently transfected with constitutive TIG1A or GRK5 expression vectors along with the indicated siRNAs for 48 h using anti-myc antibodies. β-actin expression was determined as a loading control. (B) Effects of TIG1 and GRK5 knockdown on PGE2-stimulated TOPFLASH and CREB reporter activities in transiently transfected HCT116 cells. Cells were cotransfected with indicated TIG1A, GRK5 or control expression vector, reporter plasmid and siRNA for 24 h, serum starved for 16 h, and then incubated with 10 nM PGE2 for 24 h. TOPFLASH (top panel) and CREB (bottom panel) reporter activities were determined. Representative results from triplicate samples were expressed as means ± SD. (C) Effects of TIG1 and GRK5 siRNAs on TIG1A and GRK5 expression in stable TIG1A-expressing cells. Cells were transfected with the indicated siRNAs and then incubated with or without 5 nM MFP immediately after transfection for 24 h. Expression of TIG1A and GRK5 was determined by Western blot analysis using anti-V5 and anti-GRK5 antibodies, respectively. (D) Effects of TIG1A or GRK5 knockdown on PGE2-stimulated TOPFLASH and CREB reporter activities in stable control and TIG1A expressing cells. Cells were transfected with the indicated siRNAs and then incubated with 5 nM MFP for 24 h, serum starved for 16 h, and stimulated with or without 10 nM PGE2 for 24 h. MFP was present during serum starvation and PGE2 treatment. TOPFLASH (top panel) and CREB (bottom panel) reporter activities were analyzed. (E) Effects of TIG1 and GRK5 siRNAs on TIG1A-mediated suppression of PGE2-stimulated nuclear β-catenin accumulation in stable TIG1A-expressing cells. Control and TIG1A stable cells were transfected with the indicated siRNA and then incubated with 5 nM MFP for 24 h, serum starved for 16 h, and treated with or without 10 nM PGE2 for 30 min. MFP was present during serum starvation and PGE2 treatment. Nuclear and cytosolic fractions were prepared, and subcellular distribution of nuclear and cytosolic β-catenin was determined by Western blot analysis. Normalization in the levels of nuclear and cytosolic β-catenin was described in the Materials and Methods, and the relative levels of nuclear β-catenin in control stable cells without PGE2-treatment was designated as 1.0. *, P < 0.05; **, P < 0.01; ***, P < 0.001.