Figure 6: The effects of 3-MA on self-renewal and cell cycle progression of liver CSCs. A. TEM and confocal imaging showing the presence of autophagic vesicles in GPC3+, EpCAM+, or CD133+ sub-populations isolated from PLC/PRF/5 cells. B. Western blotting for cell cycle regulators (left) and autophagic regulators (right) in GPC3+, EpCAM+, or CD133+ sub-populations isolated from PLC/PRF/5 cells. C. Spheroid formation assay showing the enrichment of self-renewing populations by serial passage in GPC3+, EpCAM+, or CD133+ sub-populations, and the significant reductions in spheroid forming cells after treatment with 3-MA in each sub-population (P <0.01 for all sub-populations). D. Western blotting showing the effects of 3-MA on the accumulation and clearance of LC3B protein in these subpopulations. E. Cell cycle analysis and F. The effects of 3-MA (low dose- 5 mM, high dose-10 mM) on cell cycle progression of the sub-populations isolated from PLC/ PRF/5 cells.