Fig. 6

Wnt7a promotes HM.4T1 sphere-forming ability via the PI3K/Akt/mTOR signaling pathway. A HM.4T1 cells were grown in 8-well Lab-Tek II chamber slides and stimulated with 100 ng/ml Wnt3a or Wnt7a. Twenty-four hour after the stimulation, nuclear translocation of β-catenin was examined by immunofluorescence. Green: β-catenin. Blue: DAPI. The scale bar indicates 20 μm. B HM.4T1 spheroids were stimulated with 100 ng/ml Wnt3a or Wnt7a for 4 days in 3D culture, and the level of β-catenin in the nucleus and cytoplasm was evaluated by western blotting. The results are presented as the mean ± SEM. Statistical significance was analyzed by two-way ANOVA with multiple comparisons. *p < 0.05, n = 3. C HM.4T1 spheroids were stimulated with 100 ng/ml Wnt7a for 24 h, and then, phosphorylation of Akt and p70S6K was evaluated by western blotting. D HM.4T1 spheroids were treated with or without 10 μM LY294002 for 1 h and then incubated for another 24 h. Phosphorylation of Akt and p70S6K or the level of c-Myc was evaluated by western blotting. To examine the effects of LY294002 on Wnt7a-induced phosphorylation of Akt and p70S6K or c-Myc expression, cells in spheroids were lysed after 6 and 24 h. E HM.4T1 spheroids were treated with or without 50 ng/ml rapamycin for 1 h and then incubated for another 24 h. Phosphorylation of p70S6K was evaluated by western blotting. To examine the effects of rapamycin on Wnt7a-induced phosphorylation of p70S6K, cells in spheroids were lysed after 6 and 24 h. F HM.4T1 spheroids were treated with DMSO or 10 μM LY294002 or 50 ng/ml rapamycin for 1 h and then incubated for up to 72 h in the presence or absence of 50 ng/ml Wnt7a. Photographs were taken at 24, 48, and 72 h. The diameter of each spheroid was measured by the Image J software. Scale bars indicate 100 μm. The results are presented as the mean ± SEM. Statistical significance was analyzed by two-way ANOVA with multiple comparisons. ****p < 0.0001, n = 6