Fig 2: Tenascin-C is elevated in SSc.(a) Tenascin-C (TNC) mRNA expression in SSc biopsy-derived microarray data sets (GSE32413); levels in individual intrinsic gene subsets (H, healthy controls; D1, diffuse 1; D2, diffuse 2; I, inflammatory) are shown as box plots spanning values from 25–75 percentile; horizontal lines represent median, maximum and minimum values. One-way analysis of variance (ANOVA) followed by Sidak's multiple comparison test. (b) RNA from SSc (n=14) and healthy adult (n=4) skin biopsies examined by real-time qPCR. Results, expressed relative to GAPDH, are means±s.d. of duplicate determinations. *P<0.05, Mann–Whitney U-test. (c) Immunofluorescence microscopy. SSc (n=18) and healthy control (n=9) skin biopsies were stained with antibodies to tenascin-C. Left panels, representative confocal immunofluorescence images; dotted lines indicate dermal–epidermal junction. Scale bar, 50 µm. Bottom, 3D plots. Right panel, quantitation of fluorescence intensity. Each point represents mean immunofluorescence intensity from four randomly selected h.p.f.'s in skin biopsies from healthy controls, and SSc biopsies classified as inflammatory (I), or diffuse-proliferative (D1/D2). Results, expressed, are means±s.d. of quadruplet determinations from a single subject. One-way ANOVA followed by Sidak's multiple comparison test. (d) Tissue levels of tenascin-C correlate with MRSS. Each dot is a single subject (red, early-stage SSc; blue, late-stage SSc). Pearson's correlation. (e) Circulating tenascin-C in SSc. Serum levels in Cohort 3 (50 SSc patients and 26 healthy controls), Cohort 4 (62 SSc patients and 10 healthy controls) and Cohort 5 (38 SSc patients and 12 healthy controls) determined by enzyme-linked immunosorbent assay. Each dot is the mean±s.d. of triplicate determinations from a single subject. Mann–Whitney U-test.

Fig 3: Tenascin-C-induced fibrotic responses are TLR4-dependent.(a–c) Human foreskin fibroblasts were incubated in media with tenascin-C (TNC; 2 µg ml-1) in the absence or presence of CLI-095 or MyD88 blocking peptide or scrambled controls for 72 h. (a,c,d) Whole-cell lysates analysed by western blotting. Representative immunoblots. S, secreted; L, lysates. Band intensities, normalized for tubulin, shown below. (b–d) Immunofluorescence (IF) microscopy using antibodies to aSMA, and DAPI. Representative images; scale bar, 50 µm. One way analysis of variance followed by Bonferroni's multiple comparison test. (d) Skin fibroblasts isolated from mice with tamoxifen-inducible fibroblast-specific TLR4 knockout (TLR4-/-) and control mice (that is, no tamoxifen) incubated in media with tenascin-C (2 µg ml-1) for 72 h. Upper panel, representative immunoblots. Lower panels, immunofluorescence; scale bar, 25 µm. Relative fluorescence intensities represent means from four randomly selected h.p.f.'s. Con, control.

Fig 4: Characterization of sEVs derived from lung cancer cell lines. Size distribution analysis and TEM image of a) HCC827, b) H1975, and c) HCC78 cell line sEVs. d) sEV surface biomarker expressions of THBS2, VCAN, and TNC by conventional ELISA. e) Nanoflow cytometry of sEVs’ expression of tetraspanins (CD9, CD63, and CD81).

Fig 5: Attenuated skin fibrosis in TNC-/- mice.Wild-type (WT) mice and TNC-/- mice in parallel received bleomycin or PBS via s.c. injections for 10 days. Lesional skin was collected at day 15 and day 24. (a) Left panels, Trichrome stain. Representative images. Scale bar, 100 µm. Arrows indicate dermis. Middle panel, dermal thickness (means±s.d. of five determinations per h.p.f. from three or five mice per group). Right panel, changes in dermal thickness comparing day 24 with 15 following initial injection. Mann–Whitney U-test. (b) mRNA levels at day 24 determined by real-time qPCR. Results, normalized with GAPDH, are means±s.d. of triplicate determinations from three mice per group. One-way analysis of variance followed by Sidak's multiple comparison test. (c) Double immunofluorescence using antibodies to phospho-Smad2 and tenascin-C. Upper panel, representative images. Scale bar, 50 µm. Inset, higher magnification; arrows indicate co-localization of tenascin-C and nuclear phospho-Smad2. Lower panel, proportion of phospho-Smad2-positive cells within the dermis determined at four randomly selected locations per h.p.f. Results are means±s.d. from three mice per group.