Extended Data Fig. 8: Characterization of the skin of Tgfbr^ΔN mice.

a, Representative images of neutrophils (green) stained for Col3a1 (red) in the skin of WT control (Cre^NEG Tgfbr^flox littermates, 5 images from 2 mice) and Tgfbr^ΔN mice (9 images from 3 mice), which we used to quantify the number of neutrophils per volume area is shown at right. Data are presented as mean values ± s.e.m., compared by two-tailed unpaired Student’s t-test. b, Histological characterization of the skin of wild-type control (n = 3) and Tgfbr^ΔN mice (n = 3) by hematoxylin-eosin staining for cell and tissue structure, Masson’s trichrome for collagen-rich structures, and Ki67 staining for dermal and epidermal proliferation. Images are quantified in the dot plot graphs. P-values were determined by a two-sided unpaired Student’s t-test. c, Examples of SHG and fibre reconstruction using CT-FIRE for estimation of fibre width in control and Tgfbr^ΔN mice also shown as distribution of widths in the histogram below (and in Extended Data Fig. 4A for control vs. iDTR mice). P-values were determined by a two-sided unpaired Student’s t-tests. d, Schematics of the atomic force microscopy (AFM) set-up used to measure the stiffness of tissue samples (left) and its quantification in the form of elastic Young’s modulus in lung, intestine and skin of Cre^NEG;Tgfbr^flox control (referred to here as WT; n = 5) and Tgfbr^ΔN littermates (n = 5 mice). Each dot represents the median Young’s modulus value calculated from ∼250 individual force–distance analysed curves per mouse. Data were compared by two-sided unpaired Student’s t-test. Right, representative height images and corresponding Young’s modulus maps of skin from control (n = 5) and Tgfbr^ΔN littermates (n = 5) acquired by AFM indentation experiments. e, TEM images of the ear skin transversal sections showing collagen fibres in the subepidermal and lower dermis regions, which were automatically segmented for analysis (coloured fibres). Yellow circles highlight large collagen fibres (>0.2 μm²) in the subepidermal region. f, Quantification of fibre size in the skin of Tgfbr^ΔN mice (19825 fibres) and Cre^NEG littermate controls (62279 fibres); data are from 2 independent experiments. P-values determined by Kruskal-Wallis non-parametric test. g, Per cent of ‘large’ fibres in the subepidermis and lower dermis from the images in f. Data are presented as mean values ± s.e.m., and p-values were determined by Kruskal-Wallis non-parametric test. h, Permeability assays in control (n = 7) and Tgfbr^ΔN mice (n = 7) measured by FITC–dextran injected either intratracheally (for lung), or by oral gavage (for gut) in Cre^NEG control (n = 8) and Tgfbr^ΔN mice (n = 8). Evans blue given intravenously to control (n = 11) and Tgfbr^ΔN mice (n = 5) and then measured in the indicated tissues. All controls here were Cre-negative Tgfbr^flox littermates. i,j, Stiffness (i) and permeability assay (j) using Evans blue in the ear skin of Mrp8^Cre; Tgfbr2^flox and Ly6G^Cre; Tgfbr2^flox mice, as well as Cre^NEG; Tgfbr2^flox littermates, or mice treated with anti-Ly6G to deplete neutrophils or isotype control antibody. The numbers of replicates are displayed in the figure. P-values determined by two-sided Student’s t-test comparing each depletion method with their controls (left panels), or one-way ANOVA with multiple comparison test (right panels). Box plots in (d,h–j) show median ± interquartile; whiskers show the range from minimum to maximum. Box plots in f show median ± interquartile; whiskers are defined with percentiles and IQR (interquartile range, P75–P25); the points outside this range are the outliers.

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