Fig. 4
ASPM is essential for proliferation of HCC cells in vitro and in vivo. A Cell viability of SNU-739 and HepG2 cells, transfected with siRNAs targeted ASPM (siASPM) or negative control (siControl), was detected by cell counting-8 kit (CCK-8). The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by one-way ANOVA. ***p < 0.001. B Cell viability of MHCC-LM3 cells, transfected with ASPM-overexpression vector or empty vector (EV), was detected by cell counting-8 kit (CCK-8). The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t test. ***p < 0.001. C Plate clone formation assay of SNU-739 and HepG2 cells, transfected with siRNAs targeted ASPM (siASPM) or negative control (siControl), was performed. Clone numbers were measured by ImageJ software. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by one-way ANOVA. ***P < 0.001. D Plate clone formation assay of MHCC-LM3 cells, transfected with ASPM-overexpressed vector or empty vector (EV), was performed. Clone numbers were measured by ImageJ software. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t test. ***p < 0.001. E Cell proliferation of HCC cells was detected by EdU staining. The scale bar is 50 μm. F Statistics of EdU cell proliferation data in Fig. 2E. The cell rate was the ratio between the number of EdU-stained cells and the total cells. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by one-way ANOVA. *p < 0.05. G The SNU-739 cells were stably infected with lentivirus expressing the shRNAs targeting ASPM and the control shRNA, respectively. And then, stable clones were injected subcutaneously into the back of 8-week-old nude mice to grow tumors. Subcutaneous tumors were stripped and photographed after injection for 5 weeks. H The mice were sacrificed after 5 weeks and tumors were removed to measure the weight. The data were presented as the mean ± SD. The significance was analyzed by one-way ANOVA. **p < 0.01, ***p < 0.001. I The tumor volume was measured and calculated by V = 0.5 × Length × Width2. The data were presented as the mean ± SD. The significance was analyzed by one-way ANOVA. **p < 0.01, ***p < 0.001. J Survival curve of mice bearing xenografts was recorded. Mice with a tumor volume greater than 600 mm3 were considered dead. K Ki-67 expression of the tumor tissues was detected by IHC and the percentage of positive cells was calculated by ImageJ IHC Profiler. The scale bar is 100 μm. The data were presented as the mean ± SD of three different fields of view at low magnification (× 40). The significance was analyzed by one-way ANOVA. ***p < 0.001. L The protein levels of ASPM and FOXM1 in subcutaneous tumors were detected by Western blot