Fig. 4
The inhibition of Notch1 signaling pathway attenuated the stemness properties of CD44v6+ LCSCs. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the expression of Notch1. The result showed that Notch1 was higher in HCC patients’ samples than that in normal samples (1.77 fold, FDR = 0.0017). b The core components of Notch1 signaling including Notch1 receptor, cleaved Notch1 (NICD), Hey1 and Hes1 were tested in CD44v6+ LCSCs and CD44v6- HCC cells by western blot in SNU-398 cell lines. Western blot showed that CD44v6+ SNU-398 cells expressed more Notch1 signaling pathway key factors. β-actin was used as a normalized control. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of Notch1 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assay showed that knockdown Notch1 decreased the migration and invasion capacity of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when Notch1 was down regulated. g and h Efficiency of tumor formation of Notch1 shRNA cells and the corresponding controls. Right flanks of mice were injected with control CD44v6+ cells while left flanks were injected with Notch1 shRNA cells. Number of injected cells: 1 × 105. n = 5. Data are expressed as mean ± SD (error bars). ** p < 0.01, ***p < 0.001 and ****p < 0.0001, t test