Asrani et al Supplementary Figure S1: Epidermal-specific mTORC1 loss-of-function models show impaired epidermal differentiation. (A) Rheb cKO P0 epidermis fails to express spinous (Keratin 1, 10) and granular layer (loricrin, filaggrin, involucrin) differentiation markers by immunofluorescence, while the basal layer is maintained or even slightly thicker than WT (p63 and Keratin 5). Scale bar= 30 µm. (B) Rheb cKO P0 epidermal protein lysates subjected to immunoblotting confirm downregulation of K1, K10, loricrin, involucrin and transglutaminase (TGM1) 1 expression compared to WT. (C) Rptor cKO P0 epidermis shows a similar but more severe phenotype compared to Rheb cKO, with near complete loss of suprabasal keratinocyte layers in the epidermis as demonstrated by basal marker p63 immunostaining. Scale bar= 30 µm. (D) Immunoblotting of Rptor cKO vs WT P0 epidermal protein lysates confirms relative downregulation of granular markers involucrin and TGM1 with slight up-regulation of basal marker keratin 14 (K14). (E) Calcium-induced differentiation is impaired in Rheb cKO cells subjected to calcium switch assay (transfer from 0.05 mM to 2 mM calcium concentration media). While WT cells up-regulate differentiation markers (K10, loricrin, filaggrin, involucrin and TGM1) in response to exposure to 2 mM calcium for 24 hours, cKO keratinocytes show an impaired response. Basal markers (K5) are unchanged in response to calcium. (F) mTORC1 inhibition by rapamycin (200 nM) in WT keratinocytes impairs expression of spinous (K10) and granular (loricrin) differentiation markers in 2 mM calcium. Phosphorylated S6 protein (p- S6) is markedly diminished by rapamycin, confirming mTORC1 inhibition. 1 Asrani et al Supplementary Figure S2: mTORC1 loss-of-function is associated with decreased desmosomal, but not adherens, junctions. (A) Decreased expression of PKP1 (punctate dots), DSP1/2, DSG1 and DSG3 in Rptor cKO compared to WT P0 epidermis by immunofluorescence. Scale bar= 60 µm. (B) Densitometry quantification of immunoblot analysis shown in main Figure 3G (n=3; error bars represent SEM; p values indicated are by Student’s T-test). (C) Rheb cKO P0 epidermal protein lysates subjected to immunoblotting confirm lower total levels of desmosomal proteins (DSP1/2, DSG1, DSC3 and PKP1). (D) Rheb cKO P0 cultured keratinocytes show normal protein expression of adherens junction components (E-cadherin, β-catenin, α-catenin), as well as adherens junctions components (afadin) and tight junction components (ZO-1) by immunoblotting. Some actin-binding proteins such as vinculin, afadin and EPLIN show decreased levels in cKO epidermis as well. (E) Rheb cKO P0 epidermis shows normal expression of adherens junction proteins by immunofluorescence. Scale bar = 30 µm. (E) Rheb cKO keratinocytes have normal membranous levels of E-cadherin, however the distribution of staining demonstrates immature adhesion zippers rather than mature adhesion bands by immunofluorescence. Scale bar = 50 µm. 2 Asrani et al Supplementary Figure S3: mTORC1 loss-of-function is associated with increased Rho kinase activity and focal adhesion formation. (A) Increased biochemical evidence of focal adhesion formation in inducible Rptor KO keratinocytes, including increased phosphorylated paxillin (p-paxillin), focal adhesion kinase (p-FAK) and p130Cas (p-p130Cas), following calcium switch. (B) Immunofluorescence for p-paxillin and phallodin staining for f-actin demonstrates relatively rare focal adhesions in WT cells compared to Rheb cKO cells where focal adhesion and actin stress fiber formation is abundant. Scale bar = 30 µm. (C) Increased phosphorylated myosin light chain (p- MLC2) in Rheb cKO P0 epidermal lysates by immunoblotting compared to WT control. (D), (E) Lysates enriched for membrane proteins from Rheb cKO and Rptor inducible KO cultured keratinocytes demonstrate increased Rho A/B/C protein at the membrane in cells with mTORC1 loss- of-function compared to WT cells, consistent with increased Rho activity. Levels of ROCK inhibitor Rho E, by contrast, are decreased in membrane lysates from Rptor KO keratinocytes. Rho A/B/C and calreticulin in (D) were immunoblotted in parallel, contemporaneously. (F) Immunoblotting for desmosomal components (DSP1/2, DSG1, PKP1) in WT and Rheb cKO cells demonstrates rescue of total levels of desmosomal proteins with ROCK inhibition using fasudil (50 µM). ROCK activity suppression is documented by decreased p-MLC2 levels. Focal adhesion marker protein p-paxillin is also upregulated in Rheb cKO keratinocytes and is sensitive to ROCK inhibition. DSP, PKP1 and total MLC2 were immunoblotted in parallel, contemporaneously with the other markers. (G) Densitometry quantification of immunoblot experiments from Figure 6C (r=3; p-values by one-way ANOVA). (H) Densitometry quantification of immunoblot experiments from Figure 6E (r=3; p-values by one-way ANOVA). (I) . Immunoblotting of Rheb WT cells treated with ROCK1 siRNA (50nm) for ROCK1 and markers of ROCK activity, desmosomal and biochemical differentiation markers. DSP (lower panel) was immunoblotted separately from the upper panel using the same biological replicate. Error bars represent SEM throughout. 3 Asrani et al Supplementary Figure S4: ALK5 levels and TGF-β signaling are increased following mTORC1 loss-of-function and TGF-β signaling is necessary and sufficient to decrease desmosome levels in keratinocytes. (A) Immunohistochemistry for c-MYC shows more diffuse expression in Rheb cKO epidermis compared to WT where it is expressed predominantly in basal cells (scale bar=30 µm). (B) Ingenuity Pathway Analysis of the TGF- β signaling pathway from differential gene expression analysis of Rheb WT vs. cKO E18.5 epidermis. Genes within our data set whose expression showed greater than 2SD differential fold change are outlined in magenta and depicted in red (signifying up-regulation) or blue (signifying down-regulation). (C) Membranous ALK5 (TGF-β receptor I) levels are increased in Rheb cKO P0 epidermis compared to WT controls by immunohistochemistry (scale bar=30 µm). (D) ALK5 levels are increased by immunoblotting in Rptor cKO P0 epidermis compared to WT control. (E) Short-term treatment of Rptorflox/flox keratinocyte cultures treated with empty adenovirus or adenoviral-cre demonstrates increased TGF-β signaling (increased p-SMAD2 levels) and increased ROCK activation by immunoblotting (increased levels of p-MYPT1 and p-MLC2) in cells with mTORC1 inactivation with TGF-β treatment. ROCK activation is also increased in WT cells in response to TGF-β. (F) Densitometry analysis of phospho/total SMAD2, 3 expression in western blots from starved and non-starved Rptorflox/flox and inducible Rptor cKO keratinocytes demonstrates significant up-regulation in Rptor cKO keratinocytes (r=4; error bars represent SEM; p values indicated are by Student’s T-test). (G) Immunofluorescence for SMAD2, 3 demonstrates increased nuclear localization in Rptorflox/flox keratinocyte cultures treated with adenoviral-cre (scale bar=50 µm). (H) Long term (30 h) TGF-β treatment of Rptorflox/flox keratinocyte cultures treated with empty adenovirus or adenoviral-cre increases p-SMAD2, 3 levels and suppresses DSP1/2, DSG1, DSG3 and PKP3 levels by immunoblotting. (I) By immunoblotting, basal p-MLC2 and p-MYPT1 levels are decreased, and desmosome proteins (DSP1/2, DSG1, DSG3) and differentiation markers (TGM1, loricrin) are increased in Rptorflox/flox keratinocyte cultures with empty vector, treated with ALK5 inhibitor SB431542. Rheb cKO keratinocytes show uniform down-regulation of desmosomal mRNA levels under low calcium (J) conditions as well as high calcium (K) conditions. ROCK inhibition with fasudil or ALK5 inhibition with SB431542 is sufficient to rescue desmosomal mRNA levels to WT levels for most of the desmosomal transcripts (r=3; error bars represent SEM; * indicates p< 0.05 by one-way ANOVA). 4 Asrani et al Supplementary Figure S5: siRNA mediated depletion of ALK5 is sufficient to inhibit ROCK signaling and rescue desmosome levels in keratinocytes with mTORC1 loss-of-function. (A) Treatment of Inducible Rptor cKO keratinocytes with ALK5 siRNA partially suppresses p-MLC2 and ALK5 levels and rescues expression of desmosomal proteins (DSP, DSG1, DSG3, PKP3) and markers of biochemical differentiation (TGM1, loricrin) by immunoblotting. (B) Treatment of Rheb cKO keratinocytes with ALK5 siRNA partially suppresses p-MLC2 and ALK5 levels and rescues desmosomal protein expression by immunoblotting. (C) Treatment of Rheb WT keratinocytes with ALK5 siRNA partially suppresses p-MLC2 and ALK5 levels and rescues expression of desmosomal proteins (DSP, DSG1, DSG3, PKP3) and markers of biochemical differentiation (involucrin) by immunoblotting. DSP and ALK5 were immunoblotted separately using the same biological replicate. (D) Treatment of Rheb cKO keratinocytes with ALK5 siRNA increases membranous desmosomal protein levels (DSP1/2 and DSG1) and cortical f-actin (by phalloidin staining) and p-MLC2 levels by immunofluorescence (left panels) (scale bar=100 µm). Quantification of DSP and DSG1 border fluorescence (right panels) from representative images (n>28 cells, r=3; p<0.001 by Mann-Whitney test). 5 Asrani et al Supplementary Figure S6: Tentative model outlining the downstream sequalae of mTORC1 loss-of-function in the epidermis. Solid lines represent pathways substantiated in this manuscript, or by previous studies (references 41,56). Dashed lines represent potential uncharacterized consequences of mTORC1 inhibition that are the subject