Fig 2: Butyrate promotes skin barrier function.a Quantification of baseline epidermal thickness in water control (CTL) or butyrate-treated mice before allergen sensitization. b Quantification of stratum corneum (SC) thickness following analysis of representative hematoxylin and eosin (H&E)-stained skin tissue before HDM allergen exposure. c Measurement of baseline dorsal transepidermal water loss (TEWL) in control or butyrate-treated mice before allergen exposure. d Expression of key components of the cornified envelope by fluorescence-activated cell sorting (FACS)-purified epidermal CD326+ keratinocytes isolated before allergen sensitization from CTL mice or butyrate-treated mice, as determined by quantitative RT-PCR. e Expression of TH2 cytokines in the skin at baseline, as determined by quantitative RT-PCR. Gene expression was normalized to ß-actin in d and e. f Representative H&E-stained skin tissue from CTL or butyrate-treated mice after 2 weeks of HDM allergen exposure and corresponding quantification of stratum corneum (SC) thickness (randomly measured 3 times per picture, 7–8 pictures per sample). Arrows represent representative sites for SC thickness measurement. Scale bars, 100 µm. g Representative immunohistochemistry-stained skin tissue from CTL or butyrate-supplemented mice after 2 weeks of HDM allergen sensitization and corresponding quantification of loricrin expression (represented by a light-to-dark brown staining). Loricrin staining was measured in the entire picture, with 4 pictures per sample. Scale bars, 100 µm. h Fold-change (2 weeks HDM-sensitized over baseline) SC thickness and loricrin expression in the skin of CTL or butyrate-exposed animals. Dotted line represents an arbitrary value for baseline fold-change. i Absolute quantities of cholesterol in the skin of naive (“baseline”) and HDM allergen-sensitized (“ADLSI”) CTL and butyrate-treated mice, as determined by ultrahigh performance supercritical fluid chromatography coupled to quadrupole time-of-flight mass spectrometry with electrospray ionisation (UHPSFC/ESI-QTOF-MSE). j Distribution of the main classes of ceramides and absolute quantities of Ester-linked-Omega-hydroxy (EO) ceramides, as determined by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionisation (UPLC/ESI-MS/MS). k Frequencies and representative histograms of FITC-dextran-positive MHCII+ dermal macrophages, Ly6cneg mono-DC, and CD11b+ cDC2 in the skin of two-time HDM allergen-sensitized control (CTL) or butyrate-treated animals after one topical administration of 100 µg FITC-dextran (10 KDa). Saline = HDM-exposed skin without FITC dextran administration (saline-treated). Frequencies and representative histograms of FITC positive cells in the skin of two-time HDM allergen-sensitized CTL or butyrate-supplemented mice after one intradermal administration of 100 µg FITC-dextran (l) or 100 µg DQ-OVA (m). Results are representative of data from 3 independent experiments in a and C, pooled from 2 independent experiments in b, d, pooled from 4 independent experiments in e, from at least three independent experiments in f–h, from one independent experiment in i, j, and from two independent experiments in k–m. All results are expressed as mean ± SEM (n = 7 CTL and n = 6 butyrate in a and c; n = 11 per group in b; n = 8/12 for CTL and n = 7/11 for butyrate in d; n = 16/23 for CTL and n = 15/23 for butyrate in e; n = 6 per group in f–h and l; n = 5 CTL “baseline”, 5 CTL “ADLSI”, 5 butyrate “baseline”, and 4 butyrate “ADLSI” in i; n = 5 CTL “baseline”, 4 CTL “ADLSI”, 5 butyrate” baseline”, and 3 butyrate “ADLSI” in j; n = 7 per group in k; n = 5 CTL and 4 butyrate in m). Statistical significance was determined with Student’s t-test (unpaired, two-tailed) in a–m, or Mann–Whitney test in e and k. *P = 0.05, **P = 0.01, ***P = 0.001, and ****P = 0.0001.

Fig 3: Influence of DHA supplementation on the skin substitutes’ morphology. (a,b) Macroscopic aspect and (c,d) histological cross-section following Masson’s trichrome staining of the skin substitutes. (b: basal layer, s: spinous layer, g: granular layer and sc: compact layers of the stratum corneum.) Scale bars: (a,b) 1 cm, (c,d,g–j) 100 µm. (e) Epidermal and (f) dermal thickness quantified from Masson’s trichrome-stained sections using ImageJ software. (g,h) Immunofluorescence staining of Lor (green) and (i,j) TGM-1 (green) with nuclei stained in blue with DAPI. (k) Western blot analysis of differentiation markers in the epidermis of skin substitutes followed by (l) quantification of signals for relative band densities using ImageJ Software. n = 6 (3 donors, 2 skin substitutes per donor, 3 measurements per skin substitute). The p-values were derived from Student’s t-tests and considered statistically significant when p < 0.05. Abbreviations: Lor, loricrin; TGM-1, transglutaminase-1; K1, keratin 1.

Fig 4: Role of cell-type-specific cyclooxygenase-2 (Cox-2) expression in Ras/p53-mediated tumor formation. a Experimental scheme. b Microscopic phenotype and histology of three-dimensional (3D) organoids with/without celecoxib treatment. c Relative 3D organoid formation was determined by the number of organoids larger than 100 µm, n = 3 independent experiments, 25 fields at high power field (HPF) per sample. Veh, vehicle control; C25, 25 µM; C50, 50 µM celecoxib. d Experimental scheme. e Ras/p53-mediated tumor incidence with/without condition Cox-2 knockout. Ptgs2wt/wt, n = 15 animals; Ptgs2flox/flox, n = 14 animals. f, g Histological phenotypes. h–k Immunoblotting of Keratin5 (Krt5), Krt6 and Loricrin (Lor) demonstrated decreased tumor susceptibility but increased differentiation status, n = 4 independent experiments. l Summary of the contribution of physiological stress factors in the susceptibility of tumor development from tumor-competent Krt15+ progenitors. Data with bar graphs are represented as mean ± SEM. Statistical significance was determined by pair-wise comparison using t test; ns = not significant, *p < 0.05, **p < 0.005. Scale bars, 100 µm. Ptgs2 prostaglandin-endoperoxide synthase 2

Fig 5: The genetic loss of epidermal Pgc-1a reduces cell growth and enhances differentiation after physiologic skin damage. (A) Schematic of cre-lox recombination to delete Pgc-1a in keratinocytes. (B) Epidermal Pgc-1a mRNA (Ppargc1a) in an n of 7–9 WT and epiPgc-1a KO mice. (C), representative images of keratin 14 (K14), keratin 10 (K10), and loricrin (Lor). (D) The UVB-induced fold-change in EdU+ epidermal cells (n = 5–6) and (E) K10 layer thickness (n = 4–7) relative to sham skin in WT and epiPgc-1a KO mice. (F), quantification of ?H2AX and thymine dimers in UVB exposed epidermis with representative images at right from an n of 5 WT and epiPgc-1a KO mice. (G) TPA-induced fold-change in EdU+ epidermal cells (n = 5–7) and (H) K10 layer thickening (n = 6–9) relative to control skin in WT and epiPgc-1a KO mice. (I) The open wound area after 9 days in an n of 7–8 WT and epiPgc-1a KO mice. All data are plotted as mean ± SEM. All data were analyzed using an unpaired two-tailed students t-test. *Significantly different (p < 0.05) from WT. Scale = 50 µm for C–H.