Figure 4.

Fused MVB-autophagosome compartment under ALP inhibition. H4 cells were treated with Veh or 20 nM Baf. (A) White arrows indicate an autophagosome (AP) (left) and a MVB (right), which are clearly distinguished under basal conditions (Veh). For an impression of the whole cytoplasm see Fig. S6A. Bar: 500 nm. (B) Baf leads to a widespread, fused AP-MVB compartment throughout the cytoplasm (also see Fig. S6A). The individual vesicles show characteristics of both APs and MVBs, because both cytoplasm and smaller vesicles resembling MVB intralumenal vesicles are present. Additionally, these structures are fused to one another. The enlarged frame on the right shows an example. Bar: 500 nm. (C) The size of the different intracellular structures is depicted. Under Veh, 11 autophagosomes (4 individual cells) and 2 MVBs were measured (2 individual cells, their definition was only possible at high magnification). Under Baf, 58 fused AP-MVB structures were measured (5 individual cells). (D) ICC analysis of the autophagy receptor protein SQSTM1, which accumulated in a punctate pattern under Baf. The number of SQSTM1 puncta per cell, analyzed by ImageJ, was increased (****p<0.0001, Veh N = 58 and Baf N = 55 individual cells were measured). Bar: 10 µm. (E) ALP flux was also analyzed by WB, for levels of SQSTM1 and autophagosome membrane marker LC3-II. The level of these markers was increased (*p = 0.026, N = 6), indicating that 20 nM Baf inhibited ALP flux.