Fig. 4: RALYL enhances stemness properties of HCC cells. | Nature Communications

Fig. 4: RALYL enhances stemness properties of HCC cells.

From: RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

Fig. 4

a Western blotting was performed to determine the expression of stemness markers in RALYL-overexpressing cells and RALYL-silencing cells. b Spheroid formation assay was used to evaluate the self-renewal ability of 8024-Vec/RALYL, MIHA-Vec/RALYL, Huh7-Control/shRALYLs and H2M-Control/shRALYLs. The numbers of primary and secondary spheroids are calculated in the bar chart below. The values represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). c Limiting dilution assay shows tumor-initiating frequency of 8024-Vec/RALYL or LO2-Vec/RALYL cells in nude mice. d qRT-PCR was used to compare fold change of expressions of stemness-related and liver differentiation-related markers in HCC cells after treating with atRA for 5 days. The values represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). e XTT assay reveals stronger chemoresistance ability of cells with higher RALYL expression compared with that of lower RALYL expression. Cells were treated with the indicated concentration of CDDP and 5-Fu for 48 h. The values represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, two-sided Student’s t-test). f Representative images of tumors induced by 8024-Vec/RALYL treated with indicated drugs (middle). The average tumor volume was expressed as mean ± SD of five mice. Representative images of RALYL IHC staining of PLC-8024-induced tumors treated with indicated reagents (lower right). Scale bar = 32 μm.

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