Impact of <i>GPC3</i> depletion on sorted EpCAM⁺ HCC cells.

<p>(A) Dual immunostaining was performed to detect the expression of EpCAM (green) and GPC3 (red). Nuclear DAPI staining is shown in the insets. Scale bar = 100 μm. (B) Real-time RT-PCR analysis of GPC3 expression in purified EpCAM⁺ cells. *Statistically significant (p<0.05). (C) Cells transduced with the indicated lentiviruses were subjected to Western blotting using anti-GPC3 and anti-tubulin (loading control) antibodies. (D) Bright–field images of non-adherent spheres on day 14 of culture. Fluorescence images are shown in the insets. Scale bar = 100 μm. (E) Number of large spheres derived from 1,000 EpCAM⁺ or EpCAM⁻ cells at day 14 of culture. *Statistically significant (p<0.05). (F) Number of secondary spheres 14 days after replating. *Statistically significant (p<0.05). (G) A proposed model for the effect of DSF in targeting tumor-initiating HCC cells.