Figure 5

Effect of TQ on β-catenin and the glycogen synthase kinase 3β (GSK-3β) pathway in RKO. Separating for membranous, cytoplasmic and nuclear protein fractions showed reduction of nuclear β-catenin and c-myc upon 30 μM TQ for up to 24 h and a short term increase of membranous and cytoplasmic β-catenin. α-tubulin is used as a total protein and cytoplasmic marker, fibrillarin as nuclear marker, and Na-K-ATPase as membranous marker (A). TQ reduces the amount of p-GSK-3β Ser9, the inactive form of the protein, at concentrations of 10 and 30 μM, shown by Western blot (B) and fluorescence microscopy (C; top panel, green: p-GSK-3β, blue: DAPI; LSM 700). Total GSK-3β protein levels are constant over time (B; C, bottom panel). Total c-myc levels were reduced at 30 μM TQ up to 24 h, an effect that was not seen for 10 μM (B). 30 μM TQ and 30 μM PI3K inhibitor LY294002 additively reduced the amount of p-GSK-3β in total protein lysates. 5 independent Western blots had been carried out and the ratio of p-GSK3-3β levels and total GSK3-3β protein has been calculated with densitometry analysis (D, E and see also Table 1 for the statistical calculations). Treatment with TQ increased total β-catenin protein but reduced the amount of c-myc protein (D). Inhibition of MEK1/2 with 30 μM UO126 reduced p-GSK-3β, while concomitant incubation with TQ did not lead to an additive reduction of p-GSK-3β (F).