Fig 6. Activation of mTORC1 signaling inhibits differentiation.

HaCaT cells (a) or NHK cells (b) were serum starved overnight and treated with increasing doses of MHY1485 or DMSO for 30 min If indicated cells were pre-treated with 100 nM Rapamycin for 30 min. Cells were harvested and protein lysates were analyzed by Western blotting with the indicated antibodies, showing that MHY1485 induces mTORC1 signaling. (c) Increasing numbers of HaCaT cells were seeded and driven into differentiation by post-confluent growth in the presence of the indicated concentrations of MHY1485. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below a quantification of 6–8 similar Western blots is shown. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01). (d-g) MHY1485 or vehicle control was topically applied to the dorsal skin of mice for 12 consecutive days with increasing doses. (d) DSFT (Double Skin Fold Thickness) was measured before the first treatment (day1) and repeated every day. Data shown are mean values from one experiment, with n = 3 mice per treatment. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison (*p ≤0.05, **p ≤0.01, *** p ≤0.001, **** p ≤0.0001). (e) Representative images of H&E-stained sections from dorsal skin of a mouse of control and MHY 1485 treated groups (scale bar, 100 μM). (f) Evaluation of histological features, including number of epidermal layers and epidermal thickness in μM. Data shown are mean values of five measurements per mouse ± SEM. Statistical significance was calculated with Mann-Whitney test (**p ≤0.01). (g) Involucrin staining of vehicle control or MHY1485 treated mice. Overview images and close-ups are shown. MHY1485 induces in vivo a psoriasis like phenotype and interferes with proper differentiation (h) Hypothetical model how mTOR serves as a switch to determine the fate of keratinocytes.