Fig. 7. Msi1 activates mTOR signaling pathway.

a Agglomerative hierarchical clustering of mTOR pathway in mouse and human orthologs and Venn diagram showing overlapping mTOR-associated genes in human EMPD and DTG mouse skin. b GSEA profile in 4 control and 4 DTG mouse samples, as well as 3 normal human skin and 4 EMPD human skin samples showing common gene enrichment set for mTOR signaling pathway. c Immunostaining against phosphorylated Akt (p-Akt), phosphorylated-S6 Ribosomal protein (pS6), phosphorylated-70 S6 (pS6k), and Hif1α in dorsal skin of control (n = 3) and DTG (n = 3) mice 48 h after continuous Doxycycline treatment. pS6 were co-stained with Krt14. d Western blotting analysis of Pten in control and DTG mouse skin 48 h after continuous Doxycycline treatment. Gapdh was used as loading control. Western blotting against PTEN in normal and EMPD human skin tissues. β-TUBULIN was used as loading control, which is identical to Supplementary information, Figs. S8a, S11j and S22e. e Diagram of WT and mutant (MUT) Msi1 consensus binding sites in Pten 3′-UTR. f Luciferase reporter activity of Pten WT and MUT 3′-UTR constructs in response to Msi1 binding. **P < 0.01. g CLIP-qPCR assay for Pten in Msi1- and IgG-immunoprecipitates from mouse primary keratinocytes. IgG represents negative control. **P < 0.01. h Histological assessment of DTG mouse dorsal skin with and without Rapamycin treatment 48 h after continuous Doxycycline treatment. Double staining of Krt10 and pS6 with Krt14 is shown. i Kaplan-Meir plots representing percent survival rates of control (n = 12), DTG (n = 12), and DTG treated with Rapamycin (n = 11) mice after continuous Doxycycline treatment. P < 0.0001. Values in the graphs represent means ± SD. Student’s t-test was used for calculating P values in f, g. Representative images are shown. Epidermis and dermis are demarcated with broken line. Scale bars, 25 μm (c, h).