FIGURE 4.

Lacritin-stimulated autophagy is calcineurin- but not mTOR-dependent and necessary for cell survival. A, HCE-T cells were either not stressed or alternatively were stressed overnight with IFNG and then treated for 15 or 120 min with 10 nm lacritin or C-25 in the absence or presence of TNF. Cell lysates were blotted for phospho-S6K or S6K (loading control). Phospho-S6K is an indicator of mTOR activity. B, quantitation of replicate experiments from A (paired t test; ns, not significant, p > 0.05). Each value plotted is the mean integrated optical density of the films (n = 3) normalized to the S6K loading control. C, HCE-T cells were stressed overnight with IFNG and then treated for 15 min with 10 nm lacritin in the presence of TNF without or with PI103, rapamycin (Rap), cyclosporin A (CsA), or rapamycin plus cyclosporin A. Viability was assessed by the MTT assay and is expressed as the -fold increase in viability, defined as the ratio of experimental viability to the control viability. Control viability was derived from cells treated with IFNG/TNF alone (ANOVA with Dunnett's post-test; **, p < 0.01). Viability with 0.1 μm cyclosporin A or 0.1 μm cyclosporin A and rapamycin treatment plus IFNG/TNF and lacritin does not significantly differ from viability of cells treated with IFNG/TNF alone (ANOVA with Dunnett's post-test; ns, not significant, p > 0.05). D, HCE-T cells stably expressing empty vector (shCtrl) or three different ATG7 shRNAs were developed. Each was stressed overnight with IFNG and then treated for 15 min with 10 nm lacritin in the presence of TNF. Viability was assessed by the MTT assay and is expressed as the -fold increase in viability as defined above (ANOVA with Dunnett's post-test; **, p < 0.01). Viability from shATG7(3) cells with IFNG/TNF and lacritin differs significantly from shCtrl cells treated with IFNG/TNF alone (t test; *, p = 0.03); thus, shATG7(1) was used in subsequent experiments. E, Western blot for ATG7 in shCtrl and shATG7 knockdown cell lines using FOXO3 as a loading control. F, shCtrl or shATG7(1) HCE-T cells were sensitized overnight with IFNG and then treated for 15 min with lacritin (10 nm) in the presence of TNF. Cells were washed and fixed, and then FOXO3 was immunolocalized. G, HCE-T cells were sensitized overnight with IFNG and then treated for different times with 10 nm lacritin (top pair) or C-25 (bottom pair) in the presence of TNF. Lysates were blotted for LC3 or tubulin (loading control). H, quantitation of G replicates plotted as indicated in B normalized to the tubulin loading control. I, HCE-T cells were sensitized overnight with IFNG and then treated for 15 min with different doses of lacritin (top pair) or C-25 (bottom pair) in the presence of TNF. Lysates were blotted for LC3 or tubulin. J, quantitation of I replicates plotted as indicated in H. H and J, two-way ANOVA (LC3-II and LC3-I); *, p < 0.05. Error bars represent S.E. Inhib, inhibitor; lacrt, lacritin; pS6K, phospho-S6K.