Figure 2: GPC3 is a critical regulator of self-renewal in HCC cells. A. Western blot and flow cytometry were used to confirm GPC3 expression in GPC3-high and GPC3-low sub-populations isolated from Huh7 and Hep40 cells. The percentage of cells that form spheroids are enriched in the GPC3-high sub-populations from both Huh7 and Hep40 cells. B. Left panel-Suppression of GPC3 expression by siRNA down-regulated spheroid formation in both Huh7 and Hep40 cells. Right panel-Effects of 3-MA (10 mM) on spheroid formation in Huh7 and Hep40 cells. C. GPC3 expression was induced in SNU-449, SNU-398, and PLC/PRF/5 cells, and GPC3 levels confirmed by Western blotting. Flow cytometry confirmed cell surface expression of GPC3. D. GPC3 induction in SNU-449, SNU-398, and PLC/PRF/5 cells promoted the percentage of cells that formed spheroids; P<0.005.