Figure 2. Itraconazole induces autophagy in glioblastoma cells. (A) U87 cells were transfected with a pEGFP-LC3 plasmid. After 36 h, cells were treated with DMSO or indicated concentrations of itraconazole for another 36 h. Formation of EGFP-LC3 puncta was visualized by fluorescence microscopy. The data are representative of 3 independent experiments. (B) U87 cells were treated with DMSO or 5 μM itraconazole for 36 h, and formation of autophagic vacuoles was examined by TEM analysis. The data are representative of 3 independent experiments. (C) U87 cells were treated with DMSO or indicated concentrations of itraconazole for 36 h. Lysosomal protease inhibitors (E64d and pepstatin A each at 10 µg/ml) were applied for 3 h at the end of treatment time of itraconazole. Conversion of LC3-I to LC3-II was examined by immunoblot. The data are representative of 3 independent experiments. (D) U87 cells were treated with DMSO or indicated concentrations of itraconazole for 36 h, and expression of SQSTM1 was determined by immunoblot. The data are representative of 3 independent experiments. Itra, itraconazole.