Fig. 4

Spermine promotes β-catenin phosphorylation and nuclear accumulation through the activation of Akt in HCC. A Western blot of the phosphorylation level and total protein expression of β-catenin in SNU-368 cells incubated with spermine. B, C Analysis of effects of spermine on the phosphorylation level (B) and total protein (C) expression of β-catenin. **p < 0.01, ***p < 0.001, one-way ANOVA, n = 5 independent experiments per group. D Effects of pretreatment with NPS2134 (10 µM), BAPTA-AM (10 µM), MK2206 (10 µM) on the spermine-induced β-catenin nuclear accumulation in SNU-368 cells after incubation with spermine for 12 h. Veh: Vehicle; NPS: NPS2134; BAP: BAPTA-AM; MK: MK2206. *p < 0.05, ***p < 0.001, one-way ANOVA, n = 5 independent experiments per group. I Immunofluorescence staining of β-catenin in SNU-368 cells. The cells were pretreated with NPS2134 (10 µM), BAPTA-AM (10 µM), MK2206 (10 µM) for 30 min before coincubation with spermine for 12 h. n = 3 independent experiments per group. F, G Effects of ectopic expression of myr-Akt1 isoform on the phosphorylation level (F) and total protein (G) expression of β-catenin in SNU-368 cells. **p < 0.01, ***p < 0.001, two-tailed unpaired t test, n = 5 independent experiments per group. H Effects of ectopic expression of myr-Akt1 isoform on β-catenin nuclear accumulation in SNU-368 cells. **p < 0.01, two-tailed unpaired t test, n = 4 independent experiments per group. I Immunofluorescence staining of β-catenin in SNU-368 cells stably expressing myr-Akt1. n = 3 independent experiments per group. J IHC staining of phosphor-Akt and β-catenin in 24 human HCC specimens. Scale bar, 50 μm. K Spearman correlation analysis of phosphor-Akt and β-catenin IHC scores. Note that the scores of some samples overlap. L Spearman correlation test between blood spermine concentration and tumor β-catenin IHC scores