FIGURE 3.

Stressed cells display enhanced autophagy that is further up-regulated by lacritin within 10 min. A, HCE-T cells were either not stressed or were stressed overnight with IFNG and then treated for 30 min without or with lacritin (10 nm) in the presence of TNF. Cell lysates were blotted for caspases (CASP) 9 and 3 or for LC3 and tubulin (loading control). LC3-I migrates more slowly than LC3-II. B, schematic diagram of autophagy with isolation membrane (left), autophagosome (center), autolysosome (right), and several autophagy mediators (LC3, ATG7, Alfy, and p62). HCE-T cells that stably express LC3 double tagged with EGFP and mCherry (22) were developed (LC3/RG cells) and used in G–M to monitor autophagic flux. Rectangles indicate the effect of pH on LC3 tags. C, HCE-T cells were sensitized overnight with IFNG and then treated for 6 h without or with TNF in the presence of no or increasing amounts of leupeptin (leu), vinblastine (vin), or wortmannin (wort). Lysates were blotted for LC3 or tubulin (loading control). D, autophagic flux is chronically enhanced by TNF stress as revealed by accumulation of LC3-II with leupeptin and vinblastine. Each value plotted is the mean integrated optical density of the films (n = 3) normalized to the tubulin loading control. E, HCE-T cells were sensitized overnight with IFNG and then treated for 30 min with lacritin (10 nm) in the presence of TNF without or with 0.1 μm leupeptin. Lysates were blotted for LC3 or tubulin (loading control). F, quantitation of replicate experiments from E (paired t test for LC3-II; *, p < 0.05) plotted as indicated in D. LC3/RG cells were sensitized overnight with IFNG and then treated for different times with 10 nm lacritin (G) or C-25 in the presence of TNF without (H) or with 0.05 μm vinblastine (vinblast) (I). Puncta per field in microscopic images were quantitated (ANOVA with Dunnett's post-test; **, p < 0.01; *, p < 0.05; ns, not significant). Representative microscopic images are shown in J–M. J, C-25 for 60 min. K, lacritin for 60 min. C-25 (L) and lacritin (M) treatment for 60 min in the presence of vinblastine (Vin; 0.05 μm) is shown. Bar, 10 μm. Error bars represent S.E.