Figure 1. Epidermal-specific mTORC1 loss-of-function models have skin barrier defects and evidence of impaired cell-cell adhesion.

(A) Immunoblotting of WT and Rheb-cKO P0 epidermis and keratinocyte cultures for markers of mTORC1 (p-S6) and mTORC2 (p-AKT, p-FOXO1, p-GSK-3β) activity. Total AKT, total FOXO1, and total GSK-3β were immunoblotted separately using the same biological replicate. (B) Immunofluorescence of WT and Rheb-cKO E18.5 epidermis for p-S6. (C) Rheb-cKO pups are smaller at P0, with thin, shiny skin (top left), and fail to exclude toluidine blue dye at E18.5 (top right), consistent with an epidermal barrier defect. Grossly visible abrasions (arrow, bottom right) and a prominent vascular pattern are seen in P0 cKO epidermis. (D) Representative histologic back skin sections from Rheb-cKO P0 pups reveal markedly thinned epidermis with loss of the upper keratinizing cell layers (top right) and grossly apparent subcorneal pustules (bottom left), filled with neutrophil debris on histology (bottom right). (E) Immunoblotting of WT and Rptor-cKO P0 epidermis and WT and inducible Rptor-cKO keratinocyte cultures for markers of mTORC1 (p-S6) and mTORC2 (p-AKT) activity. (F) Rptor-cKO pups have visible epidermal blisters at P0 (right, arrow). (G) Rptor-cKO pups have a thinned epidermis by histology (right half of left panel, H&E staining) with visible blistering (denoted by an asterisk). The epidermal blisters in Rptor-cKO P0 pups are suprabasal as demonstrated by p63 immunostaining that labels basal cells left behind after superficial epidermal layers (right panel, arrow) are shed. Scale bars: 30 μm throughout.