Figure 3: GPC3 mediates self-renewal via induction of autophagy. A. Immunofluorescence of LC3B (red), GPC3 (green) and DAPI (blue) showing the presence of autophagic vesicles in SNU-449, SNU-398, and PLC/PRF/5 cells after GPC3 induction. B. Western blotting for autophagy-related genes upon induction of GPC3 in SNU-449 cells. C. Induction of GPC3 promoted accumulation and clearance of LC3B-II protein in SNU-449 cells (in ‘No 3-MA’ panel). 3-MA (10 mM) resulted in accumulation (without clearance) of LC3B-II protein in both un-induced SNU-449 cells (GPC3-null) and doxycycline-induced SNU-449 cells (GPC3 positive). D. Treatment of cell lines over-expressing GPC3 with 3-MA down-regulated spheroid formation (P<0.005).