Fig. 7
From: Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD
I1061T NPC1 traffics to autophagosomes in vivo. a Seven-week-old I1061T/I1061T-Npc1, GFP-LC3 or wildtype-Npc1 (WT), GFP-LC3 mice were treated with vinblastine for 2 h. NPC1 (green) and LC3-GFP (red) in hepatocytes were visualized by confocal microscopy. Nuclei were stained with DAPI (blue). Due to expression differences and to prevent overexposure, WT and I1061T were imaged at different exposures. Scale bar = 25 μm. b Whole liver homogenates (Hom) from 7-week-old WT or I1061T-Npc1 mice, or fractions enriched for cytosol (Cyt), ER, autophagosomes (APG), autolysosomes (AUT), or lysosomes (Lys) were analyzed by western blot. Blots were probed for LC3 (autophagic compartments), calreticulin (ER), or glucocerebrosidase (GBA; lysosomes). NPC1 band intensity was normalized to total protein as determined by Ponceau S stain (see Supplementary Fig. 8a). c Left: Electron micrographs of livers from WT and I1061T-Npc1 mice. Yellow arrows: autophagosomes (APG); red arrows: autolysosomes (AUT). Inserts: Representative images of APG and AUT. * indicates ER inside APG. Graphs: Average number of APG and AUT per field (left) and average number of autophagosomes containing ER or other cargo (right) were calculated by morphometric analysis of N = 38 fields from 3 mice. Scale bar = 1 μm. d Electron micrographs of livers from WT and I1061T-Npc1 mice. Arrows indicate ER. Right: Average ER diameter calculated by morphometric analysis of N = 38 fields from 3 mice. Scale bar = 1 μm. Data are mean ± s.e.m. Student’s t-test (c); average AUT/field t = 3.2, average APG-ER t = 1.8; ER diameter t = 6. *P < 0.01, ***P < 0.0001. Scale bar: white—1 µm, black—0.2 µm
