Figure 6. Western blot analysis of EMT markers and Hh signaling activity in Huh-7 and HLE subpopulations.

A. Western blot image of E-cadherin and snail in Huh-7 and HLE subpopulations. β-actin was used as a loading control. B. Zeb1 levels in various hepatoma subpopulations. TATA box binding protein was used as a nuclear protein loading control. C. PTCH1 levels in the membrane fraction of Huh-7 subpopulations were determined by Western blot analysis with integrin β1 as a loading control. D. GLI2 levels in nuclear fractions of Huh-7 and HLE subpopulations. E. Hh signaling activity after transfection with GLI-lux plasmid and mutated plasmid (GLI-Lux-M) in the presence or absence of cyclopamine (5 μM CPM). Luciferase activity was determined one day after transfection of the plasmids. ** p <0.01 compared to Huh-7 CD133⁺/EpCAM⁺ subpopulation. Δ, ΔΔ p <0.05 and 0.01 compared to GLI2-Lux+DMSO. DMSO was used to dissolve CPM, and included in transfection controls. F. Inhibition of cell proliferation by cyclopamine in Huh-7 CD133⁺/EpCAM⁺ subpopulation. Cell proliferation was determined after transfection with the GLI-Lux plasmid using the WST-1 reagent and expressed by optical density per 100 μg protein. Fugene-6 was included as a transfection reagent control.