Modulation of β1 integrin in 3D cultures of Dcn^−/− fibroblasts at day 6 by the DS proteoglycan decorin.

<p>(A) Immunfluorescence staining of Dcn^−/− fibroblasts for β1 integrin in µ-slide VI after treatment with either decorin or decorin core (core), visualized by Cy3 conjugated secondary antibody (red). Nuclei were stained with DAPI (blue). Images show merged z-axis layers of the complete 3D matrix (bar = 100 µm). (B) Representative Western blot of 3D cultures extracts for β1 integrin and the respective control GAPDH (lower panel). (C) Quantification of β1 integrin signals shown in B in grey-scale values of Western blot signals and normalized to GAPDH as loading control. Student’s <i>t</i> test (unpaired) revealed a significant difference for decorin treated cells compared to controls. Data represent 3 independent experiments and are expressed as mean ± SD (*, p<0.05). (D) Quantification of β1 integrin mRNA-levels by qRT-PCR. CT values were normalized to reference genes as described in Materials & Methods. Data represent 3 independent experiments and are expressed as mean ± SD (*, p<0.05).</p

Collagen VI distribution in fibrotic skin lesions of wild type mice.

<p>Mice were treated for 4 weeks with bleomycin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105686#s4" target="_blank">Methods</a>. NaCl injection served as control. Frozen sections were incubated with affinity purified antibodies against the collagen VI α3 (green), α5 (green) or α6 (green) chains. The sections stained for the α5 and α6 chains were co-stained with an antibody against the endothelial marker CD31 (red). Nuclei were stained with DAPI (blue). Bar, 50 µm.</p

Detection of vimentin <i>in vivo</i> in Dcn^−/− and wild-type mouse skin and <i>in vitro</i>.

<p>(A) Western blot for vimentin (57 kDa) with two different skin extracts of adult wild-type and Dcn^−/− mice (upper panel). Coomassie gel was used as loading control and shows the corresponding protein extracts (lower panel). (B) Quantification of Western blots, where the vimentin signal was normalized to the Coomassie gel staining (n = 4; **, p<0.01). (C) Dcn^−/− fibroblasts were cultured for 3, 6 and 14 days in the presence of ascorbate-2-phosphate and treated with decorin or decorin core (core) and the respective controls. Protein extracts were immunoblotted with antibodies to detect vimentin. Coomassie gel was used as loading control (lower panel, exemplary). (D) Quantification of vimentin protein expression at day 3, 6 and 14. Western blot signals were normalized to Coomassie staining. Data represent 3 independent experiments and are expressed as mean ± SD (*, p<0.05).</p

Analysis of nuclear morphology index.

<p>(A) Nuclei in 3D cultures of Dcn^−/− fibroblasts treated with either decorin or decorin core (core) as well as 3D cultures of wild-type fibroblasts were stained with DAPI (blue) at day 6 in µ-slide VI. (B) Length and width of nuclei were measured using measureIT 5.1 (Olympus) and the length:width ratio was determined. The value of wild-type nuclei was set at 1 and corresponds to a more circular shape of the nuclei. Student’s <i>t</i> test (unpaired) revealed significant differences between the Dcn^−/− fibroblasts compared to wild-type and the decorin or decorin core treated samples. Data represent 3 independent experiments and are expressed as mean ± SD (for each condition, a total of 50 nuclei were measured; *, p<0.05; **, p<0.01).</p

Expression of vimentin in 3D cultures of Dcn^−/− fibroblasts treated with either decorin or decorin core (core) at day 6.

<p>Immunofluorescence staining of vimentin (A), decorin (C) and collagen type I (D) in µ-slide VI, visualized by Alexa 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Images show merged z-axis layers of the complete 3D matrix. B: Quantification of vimentin immunofluorescence signal in merged layers normalized to cell number (nuclei) per image. Data represent 5 independent experiments and are expressed as mean ± SD (for each condition and experiments, 15 images were evaluated; **, p<0.01). Bar = 100 µm.</p

Analysis of the collagen VI α3 chain in primary fibroblast cultures from wild type and <i>Col6a1</i> null mouse skin.

<p>Cells were isolated from newborn wild type and <i>Col6a1</i> null mice and cultured for 4 days. (a) Immunostaining for the collagen VI α3 chain (green) and the endoplasmic reticulum marker PDI (red). Nuclei were stained with DAPI (blue). Bar, 100 µm. (b) Immunoblot analysis of collagen VI assembly in cell lysates (C) and supernatants (S). Cells were lysed with SDS-PAGE sample buffer, samples treated with 2 M urea and separated under non-reducing conditions on an agarose-polyacrylamide (0.5%/2.4%) composite gel. Immunoblots were developed with an antibody against the collagen VI α3 chain. (*) indicates the mobility of the single α3 chain, (#) indicates α3 chain dimers. (c) Higher magnification from (a). Bar, 50 µm.</p

Analysis of collagen fibrils in wounds of wild type and <i>Col6a1</i> null mice and of basement membranes and tensile strength in skin of <i>Col6a1</i> null mice.

<p>(a) Picrosirius red staining of day 7 wounds. d =  dermis, e =  epidermis, g =  granulation tissue, st =  scar tissue. Bar, 200 µm. (b–g) Transmission electron microscopy of day 7 wounds (b–d) and of blood vessels (e), nerves (f) and muscle (g) in unwounded <i>Col6a1</i> null skin. Arrows indicate duplicated basement membranes. (h, i) Quantification of the distance between fibrils (wt = 903; <i>Col6a1</i> null  = 1776) (h) and of fibril diameter (wt = 890; <i>Col6a1</i> null  = 1091) (i) in areas from (b) and (c), (two electron micrographs from two animals per genotype were used for quantification). Load (j) and stress (k) are significantly reduced in unwounded <i>Col6a1</i> null skin. *<0.05, **<0.005, ***<0.0005. Bar, 250 nm.</p

Collagen VI α3 expression during skin wound healing.

<p>Frozen sections of wounds of wild type and <i>Col6a1</i> null mice at 4, 7, 10 and 14 days after wounding were incubated with an affinity purified antibody against the collagen VI α3 chain followed by an Alexa 546 labeled secondary antibody (red). Nuclei were stained with DAPI (blue). d =  days after wounding. d =  dermis, e =  epidermis, g =  granulation tissue, st =  scar tissue. Bar, 200 µm.</p

Cell surface proton concentration and activity of β1 integrin in Dcn^−/− fibroblasts is altered by decorin treatment.

<p>(A) Determination of the proton concentration on the cell surface after treatment with 0.3125 µg/ml decorin and decorin core (core). Dcn<i>^−/−</i> fibroblasts were cultured over night on collagen type I coated wells as described in Materials and Methods (Decorin n = 22 cells; core n = 33 cells; *, p<0.05, n.s. = not significant). (B) Western blot with β1 integrin antibody 9EG7 that recognizes the active form of β1 integrin of Dcn^−/− fibroblasts cultured for 6 days in the presence of ascorbate-2-phosphate and decorin or decorin core (core) (upper panel). The quantification of the Western blot for active β1 integrin is normalised to total protein stain (lower blot) (3 independent experiments; *, p<0.05, paired t’test). (C) Western blot for vimentin of Dcn^−/− fibroblasts cultured for 6 days in the presence of ascorbate-2-phosphate and decorin or decorin core (core). 6 h prior to harvesting the cells were treated with a β1 integrin blocking antibody and analysed for vimentin (upper panel). The loading control GAPDH is shown in the lower panel. (D) Quantification of the Western blot for vimentin normalised to GAPDH. Data represent 3 independent experiments and are expressed as mean ± SD.</p

Influence of decorin on the a2 integrin subunit.

<p>(A) α2 integrin subunit expression in Dcn^−/− fibroblasts cultured for 6 days in the presence of decorin or decorin core and the GAPDH control (lower panel). (B) Quantification of α2 integrin Western blot signals normalised to GAPDH (3 independent experiments; *, p<0.05). (C) Analysis of α2^−/− fibroblasts compared to wild-type by Western blot and the quantification of vimentin normalised to total protein.</p