Figure 3.

ATG4B-targeted compounds inhibit starvation-induced autophagy in Saos-2 cells. Saos-2 cells stably expressing GFP-LC3B were incubated under fed and starved conditions or under starvation conditions (St) in the presence of ATG4B-targeted compounds. After 4 h, the cells were fixed and the GFP-LC3B labeled AVs visualized by fluorescence microscopy (arrows). Fed Saos-2 cells sustained in nutrient-rich medium contained few AVs (A). Nutrient-starved cells contained numerous AVs (B), that were absent when treated with NSC185058 (C) or NSC377071 (D). Scale bar (A–D): 10 μm. (E) NSC185058 and NSC377071 inhibited autophagy in a dose response fashion. (F to I) Saos-2 cells were incubated under fed (G) or starved conditions (H) or starved conditions with NSC185058 (I) or NSC377071 (image not shown). At 4 h, the cells were fixed, processed for CMPase cytochemistry, and AVs (arrows) visualized by electron microscopy. The insets contain higher magnifications of representative AVs. Scale bar (G–I and insets H and I): 1 μm. (F) The fractional volume of AVs was quantified using morphometric methods described in Materials and Methods. The values represent the mean ± SEM (n = 3). ***P < 0.001.