Propranolol and 2DG combined treatment results in massive accumulation of autophagosomes. PC3 cells were treated with 2DG (10 mM), propranolol (P) (100 µM), or both together, and were collected at the indicated times. (a,b) The levels of LC3-II and p62 were investigated by immune-blotting after 24 h or 48 h of treatment and normalized to Erk1/2 used as protein loading control. LC3-II and p62 further accumulate when the two drugs were added simultaneously, highlighting a dramatic accumulation of autophagosomes. Results are expressed as fold increase as compared to the control condition. (c–h) PC3 cells were collected after 24 h of treatments and prepared for electron microscopy analysis as detailed in “Materials and Methods”. Representative images of a cell in control condition (c,g), after exposure to P (d), to 2DG (e) or to the combined treatment (f,h) show an accumulation of autophagosomes in 2DG + P condition (see white arrows, c to f scale bar = 1 µm, (g,h) scale bars = 5 µm).