Figure 1.

Starvation-induced autophagy requires ATG4B. Saos-2 cell lines stably expressing GFP-LC3B were treated with lentiviral nonspecific “scrambled” shRNA (shCon) or ATG4B-directed shRNA (shATG4B). Stable lines expressing shCon-Saos (A and B) and shATG4B-Saos-2 (C and D) were incubated in medium enriched for amino acids and serum (A and C) or starved for amino acids and serum (B and D) for 4 h and GFP-LC3B visualized by fluorescence microscopy. GFP-LC3B labeled AVs were present in starved cells that contained ATG4B, but absent from cells lacking ATG4B. Scale bar (A–D): 10 μm. (E) Protein degradation in shCon-Saos-2 and shATG4B-Saos-2 cells was measured under fed and starved conditions as described in Materials and Methods. (F–H) shCon-Saos-2 and shATG4B-Saos-2 cells were incubated under fed and starved (G and H) conditions and the fractional volumes of AVs quantified from electron micrographs randomly selected from 3 independent experiments using morphometric methods described in Materials and Methods (F). Autophagic vacuoles (arrows) were identified by their pleomorphic structure and heterogeneous and/or CMPase (electron dense reaction product) content. The insets contain higher magnifications of representative AVs. Scale bar (G–H and insets): 1 μm. The values represent the mean ± SEM *P < 0.05; ***P < 0.001.