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. 2014 Nov 14;11(1):88–99. doi: 10.4161/15548627.2014.984277

Figure 5.

Figure 5.

Osmotic imbalances are sufficient to induce LC3 lipidation onto lysosomal compartments in a V-ATPase-dependent manner. (A and B) Confocal images of GFP-LC3 and LAMP1-RFP from time-lapse microscopy of (A) entotic corpse vacuoles or (B) lysosomes in MCF10A cells treated with hypotonic media. Arrow indicates GFP-LC3 lipidation onto entotic corpse vacuole. Bar = 2 μm. (C) Western blot analysis of LC3 in MCF10A cells cultured in control and hypotonic media for 1 h. Quantification of LC3-II/LC3-I graphed below. (D) Confocal images GFP-LC3 in wild-type, atg13−/− and atg5−/− MEFs cultured in control or hypotonic media for 30 min. Arrows indicate GFP-LC3 on vacuoles. Bar = 4 μm. (E) Quantification of LAMP1-GFP vesicle size in MCF10A cells under control or hypotonic conditions with or without Baf (100 nM); NS, not significant. (F) Quantification of hypo-osmotic induced LC3 lipidation onto LAMP1-positive entotic corpse vacuoles with or without Baf (100 nM). Data are mean ± SEM from 3 independent experiments; P < 0.01 *. (G) Western blot of LC3 in MCF10A cells in control or hypotonic media with or without Baf (100 nM). Quantification of LC3-II/LC3-I graphed below. See also Figure S5; Movie S3 and Movie S4.