Figure 4. ERC targeted β-catenin translocates to the nucleus in the presence of Wnt activator or GSK-3β inhibitors.

(A) A431 cells were serum starved for 18 h, incubated in PBS or 1 µM LPA for indicated time, then treated with either 40 mM KCl or LiCl for 5 min, immunostained for β-catenin using a monoclonal IgG antibody, and imaged with a Zeiss LSM-510 confocal microscope (40× Plan-NEOFLUAR objective; NA 1.3; scale bar = 50 µm). Cells treated with both LPA and LiCl displayed concentrated β-catenin accumulation in their nuclei (especially at 90 min when AJ dissociation occurs); whereas, those cells pretreated with PBS and KCl/LiCl together or LPA and KCl together (control salt) displayed no nuclear accumulation. (B) Cells were serum starved, treated with PBS or 1 µM LPA for 1.5 h, subsequently incubated in the presence or absence of 40 mM LiCl for indicated time, immunostained for β-catenin, and imaged. LPA-treated cells accumulated β-catenin at the ERC and showed higher catenin nuclear translocation than control upon treatment with LiCl, particularly at the 30 min time point. (C) Cells were serum-starved, stimulated with LPA or PBS for 1 h, pretreated with LiCl as indicated, and further incubated with an antibody against β-catenin and Alexafluor 647-transferrin for 30 min. The lateral images of cells were reconstituted from confocal z-sectioned images (β-catenin, red; transferrin, green). This view captures β-catenin co-distribution with transferrin in the perinuclear ERC when cells were stimulated with LPA alone (arrow head). Cells treated with both LPA and LiCl displayed accumulation in the central nuclear region (open arrow). In both cases, AJ β-catenin levels (arrows) were reduced. (D) Serum-starved cells were stimulated with PBS or LPA±LiCl as indicated, and the nuclear and cytoplasmic fractions were isolated. Western blot using anti-β-catenin (top panel) and anti-PCNA monoclonal antibodies (lower panel) was performed according to the Odyssey Infrared Imaging System. LiCl-LPA costimulation increased β-catenin level normalized by PCNA band intensity, compared to LPA or LiCl alone (N = 3). (E) Cells were stimulated by PBS or LPA for 1.5 h, incubated in the presence or absence of 1 µM GSK-3β inhibitor VIII or XIII for 30 min, and immunostained for β-catenin. Addition of specific GSK inhibitors resulted in β-catenin translocation to the nucleus in LPA-treated, but not control cells (N = 3).