Figure 1.
Detection of vimentin in vivo in Dcn−/− and wild-type mouse skin and in vitro.
(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).
Figure 2.
Expression of vimentin in 3D cultures of Dcn−/− fibroblasts treated with either decorin or decorin core (core) at day 6.
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.
Figure 3.
Analysis of nuclear morphology index.
(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 t 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).
Figure 4.
Modulation of β1 integrin in 3D cultures of Dcn−/− fibroblasts at day 6 by the DS proteoglycan decorin.
(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 t 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).
Figure 5.
Influence of decorin on the a2 integrin subunit.
(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.
Figure 6.
Cell surface proton concentration and activity of β1 integrin in Dcn−/− fibroblasts is altered by decorin treatment.
(A) Determination of the proton concentration on the cell surface after treatment with 0.3125 µg/ml decorin and decorin core (core). Dcn−/− 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.
Figure 7.
Analysis of vimentin synthesis and degradation in 3D cultures of Dcn−/− fibroblasts at day 6 treated with either decorin or decorin core (core).
(A) Quantification of vimentin mRNA expression with qRT-PCR (n = 3 independent experiments; *, p<0.05). CT values were normalized to reference genes. (B) Immuno fluorescence staining for phospho-vimentin P-Ser72 in µ-slide VI visualized by Alexa 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Images show merged of z-axis layers of the complete 3D matrix (bar = 100 µm). (C) Quantification of phospho-vimentin immunofluorescence signal in merged layers normalized to cell number (nuclei) per image and related to total amount of vimentin (Fig. 5) (n = 3 independent experiments; for each condition, 15 images were measured). Student’s t test (unpaired) revealed differences between the core-treated and the control cells. Data are expressed as mean ± SD (n = 3 independent experiments; *, p<0.05). (D) Vimentin immunoprecipitation followed by a Western blot for total sulfation (P-Ser/Thr/Tyr). Coomassie gel for total protein is shown in the lower panel. (E) Quantification of P-vimentin normalized to total protein amounts. Data represent 3 independent experiments and are expressed as mean ± SD. (F) Dcn−/−3D cultures were metabolically labeled for 12 h with 100 µCi/ml 35S-Methionine/35S-Cysteine followed by fluorography. Immunoprecipitation with vimentin antibody showed the de novo synthesis is increased by decorin protein core treatment. (G) Quantification of vimentin de novo synthesis. Data are expressed as mean ± SD (n = 3 independent experiments; *, p<0.05).