Western blot analysis showed that treatment with the Gli inhibitor GANT61 induced the accumulation of BVD-523 clinical trial LC3II in all three HCC cell lines (Fig. 2A). Treatment with the Smo inhibitor GDC-0449 also increased the LC3II level, albeit the effect was less prominent compared to GANT61. In contrast, activation of Hh signaling by its ligand (Shh) and agonists (SAG or Pur) decreased the level of LC3II. In addition to LC3II western blot, we further used fluorescence microscopy to determine the redistribution of GFP-LC3 (LC3 is a mammalian homolog of yeast Atg8 and is
normally expressed in a diffuse pattern in resting cells; during autophagy, autophagosomes engulf bulk cytoplasmic constituents including proteins and organelles, and along this process, the cytosolic form of LC3 [LC3I] is conjugated to phosphatidylethanolamine to form LC3II, which is recruited to autophagosomal membranes resulting in a more punctate distribution pattern). As shown in Fig. 2B, GANT61 treatment induced GFP-LC3
dot redistribution from a diffuse pattern to a punctate cytoplasmic pattern (GFP-LC3 puncta) in all three HCC cell lines. The Smo inhibitor GDC-0449 also induced GFP-LC3 puncta formation, although the effect was slightly less prominent compared to GANT61. These findings indicate that inhibition of Hh signaling induces autophagy and that the Gli inhibitor GANT61 is a potent agent that induces autophagy. Although GANT61-induced autophagy is observed in all three HCC cell lines, the effect is most prominent Epigenetics Compound Library purchase in Huh7 cells (Fig. 2A). To further document the effect of GANT61 on autophagy, we performed dose-dependent experiments in Huh7 cells (the cells were treated with GANT61 at 5 μM, 10 μM, or 20 μM concentration for 24 and 48 hours; quantitative assessment for the ratio of LC3II to LC3I was used as the primary indicator of autophagy induction). As shown in Fig. 2C, GANT61-induced LC3II accumulation in a dose-dependent manner. Increased detection of autophagic markers, such as LC3II accumulation and GFP-LC3 redistribution,
can result from either increased autophagosome formation or inhibition of ongoing autophagosomal maturation. To delineate these possibilities, the cells were pretreated with 3-methyladenine (3-MA, a classical inhibitor of autophagy at the sequestration stage) or E-64d/pepstatin A (lysosomal protease inhibitors that block autophagolysosomal MCE degradation) prior to GANT61 treatment. As shown in Fig. 2D, 3-MA treatment abolished GANT61-induced LC3-II formation, whereas E-64d/pepstatin A treatment augmented GANT61-induced LC3-II accumulation. The protein, p62/SQSTM1, binds directly to LC3, incorporates into the completed autophagosomes, and becomes degraded in autolysosomes. In our system we observed that GANT61 treatment decreased the level of p62 in Huh7 cells and the effect was reversed by 3-MA and E-64d/pepstatin A. Taken together, these findings suggest that the Gli inhibitor GANT61 enhanced autophagic flux.