Figure 6.

(a) HeLa cells were treated with or without 100 mM trehalose and rapamycin for 24 hours in the presence or absence of E64d and pepstatin. The level of LC3 protein was determined by western blotting, and then ratios of LC3-II/LC3-I was calculated. (b) Then, activities of mTORC1, ULK1 and S6 ribosomal protein were determined by western blotting. β-actin served as a loading control. HeLa cells were treated with or without sucrose or raffinose in the presence or absence of E64d and pepstatin. (c) Western blotting was performed to determine the conversion of LC3-I to LC3-II and activities of mTORC1, mTORC2, ULK1 and S6 ribosomal protein. Additionally, the cells were imaged for AO staining assay using a laser scanning confocal microscope (d,e). The means of red/green fluorescence ratios for individual cells were determined in three independent experiments of AO staining. The data were shown as means ± SD of three independent experiments, and representative figures were shown. Bars = 20 μm.