FIGURE 8.

Lacritin stimulated FOXO1-ATG7 and FOXO3-ATG101 coupling. A, HCE-T cells were either not stressed or stressed overnight with IFNG and treated for different times with TNF. FOXO1 was immunoprecipitated (IP) from cell lysates, and the immunoprecipitated material was blotted for acetyl-lysine, ATG7, or FOXO1 (loading control). B, quantitation from replicate blots (ANOVA with Dunnett's post-test; *, p < 0.05; **, p < 0.01). Each value plotted is the mean integrated optical density of the films (n = 3) normalized to the FOXO1 loading control. C, same as A in which stressed cells were also treated with 10 nm lacritin or C-25. D, quantitation from replicate blots (ANOVA with Dunnett's post-test; *, p < 0.05; **, p < 0.01) plotted as indicated in B. E, replicate blot of the 15-min time point from C without or with lacritin. F, quantitation of replicate blots from E plotted as indicated in B (t test; **, p < 0.01). G, HCE-T cells were either not stressed or stressed overnight with IFNG and treated for different times with TNF. FOXO3 was immunoprecipitated from cell lysates, and the immunoprecipitate was blotted for acetyl-lysine, ATG7, or FOXO3 (loading control). H, quantitation from replicate blots (ANOVA; **, p < 0.01) plotted as indicated in B but normalized to the FOXO3 loading control. I, HCE-T cells were either not stressed or stressed overnight with IFNG and treated for different times with 10 nm lacritin in the presence of TNF. FOXO3 was immunoprecipitated from cell lysates. Immunoprecipitated material was blotted for ATG101 or FOXO3 (loading control). J, replicate blot of the 15-min time point from I with 10 nm lacritin, C-25, or lysate alone. K, quantitation from replicate blots (ANOVA with Dunnett's post-test; *, p < 0.05; **, p < 0.01) plotted as indicated in H. Error bars represent S.E. lacrt, lacritin; ac-lys, acetyl-lysine.