Figure 1

L-leucine mitigates dysfunction of autophagy in NPC (−/−) cells. (A,B) Serum starvation induces an enlargement of lysosomes. NPC (−/−) cells were triply stained with lysotracker to visualize lysosomes (red), fluorescent phalloidin to label F-actin (green) and DAPI to mark DNA (blue). When kept in a serum-free medium (SFM) for 8 h, NPC (−/−) cells developed a larger number of enlarged autolysosomes (arrows in panel A) than did the cells maintained in a control medium (control; B). Scale bar; 10 µm. (C,D) EM observations reveal ultrastructural abnormalities of intracellular organelles in NPC (−/−) cells exposed to serum starvation. The identical field of NPC (−/−) cells kept in SFM was observed by fluorescence microscopy in panel (C) and by electron microscopy in panel (D). Scale bar: 20 µm. The cells indicated by box-1 and box-2 in (D) are observed at higher magnifications in panels (G,H) for box-1 and panels (I,J) for box-2. Scale bar: 2 µm (G,I), 1 µm (H,J). (E,F) An NPC (−/−) cell kept in a normal medium. The boxed region in (E) is enlarged in (F). (G,J) Higher magnification images of serum-starved NPC (−/−) cells. Autolysosomes (arrow), mitochondria (arrowhead), lamellar inclusions (asterisks) and skein-like inclusions (double arrow) are highlighted. (K–N) L-leucine supplementation rescues autophagy defects in serum-starved NPC (−/−) cells. Images of serum-starved NPC (−/−) cells stained for p62 (green) and DAPI (blue) are shown before (K) and after (L) L-leucine administration. The relative areas containing p62 immunoreactive materials in a cell (%) were analyzed by the imaging program MetaMorph (M). The areas containing lysotracker signals in a cell (%) are shown in (N). Cells were analyzed in 13 (p62) or 25 (lysosome) fields chosen at random for each condition (Dunnet post-test *p < 0.05). NPC (−/−) cells were serum-starved for 8 h, followed by a 16-h incubation with 10 mM L-leucine dissolved in a serum-free DMEM. SFM: cells in a serum free medium; SFM/add L: cells treated with L-leucine. (O) Western blot analysis of the indicated proteins in NPC (−/−) cells sampled at different time points (in h) before (0), and after (2–8) the replacement of a normal culture medium with a serum-free medium (SFM) and 16 h after the treatment with L-leucine (dissolved in a serum-free DMEM).