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

<p>(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 <i>t</i> 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).