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. 2016 Dec 1;8(2):2604–2616. doi: 10.18632/oncotarget.13733

Figure 6. YB-1 promote HCC initiating cell properties and population.

Figure 6

(A) Stemness genes were downregulated and the differentiated gene was upregulated in YB-1 KD HCC cells. Stemness genes, Nanog, Oct4 and cMyc were decreased in YB-1 KD HuH7 cells. (B) Hepatic maturation marker gene, albumin, was up-regulated in YB-1 KD HuH7 cells. Relative expression of genes in HCC cells was analyzed by real-time PCR. Expression levels were normalized to that of GAPDH. Each bar represents the means of three determinations ± SD. *p < 0.05 and **p < 0.01 among the indicated groups. (C) Numbers of side-population (SP) cells were decreased in YB-1 KD HuH7 cells. The cells were detached, labeled with the Hoechst 33342 in the presence or absence of 50 μM verapamil and then analyzed by flow cytometry (BD Aria III). The SP cells disappeared in the presence of verapamil (lower panel). (D) Numbers of EpCAM positive cells were decreased in YB-1 KD HuH7 cells. The expression of HCC stem cell marker, EpCAM, bound with anti-EpCAM conjugated FITC antibodies was analyzed by flow cytometry (BD canto II). (E) HCC initiating cells frequency was declined in YB-1 KD HuH7 cells. Limiting dilution analysis of sphere formation was conducted to estimate the frequency of HCC initiating cells by fitting the single-hit Poisson model to the limiting-dilution data. The frequency of HCC stem cells was calculated using the extreme limiting dilution analysis platform.