Glycolysis blockade by 2DG induced an increase of autophagy flux in PC3 cells. PC3 cells were treated or not with 10 mM 2DG for 24 or 48 hours. (a) 2DG treatment induces an increase of autophagy as showed by LC3-II and p62 accumulation both at 24 and 48 hours. (b) Cells were treated or not with 2DG and with (+E64d), or without, 10 μg/ml of the cathepsin inhibitor E64d for 48 h. Blocking the lysosomal proteases with E64d further enhances the 2DG-dependent accumulation of LC3-II and p62. Erk1/2 was used as protein loading control. (c) Autophagy flux was investigated by the transient overexpression of a LC3-eGFP-mCherry construct combined, or not, with 10 mM 2DG treatment for 24 or 48 h. Representative fluorescent microscopy photographs are shown (scale bars = 10 µm). No accumulation of autophagosomes is observed in 2DG condition.