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. 2011 Sep;31(18):3773–3789. doi: 10.1128/MCB.05263-11

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Fig. 7. — Decreased collagen synthesis in vimentin knockout cells. (A) Expression of type I collagen in embryonic fibroblasts from wt (Vim^+/+) and vimentin knockout (Vim^−/−) mice. (Top) Representative Western blot of cellular (CELL; lanes 1 and 2) and secreted (SEC; lanes 3 and 4) levels of collagen (I) α1 and α2 polypeptides of Vim^+/+ cells (lanes 1 and 3) and Vim^−/− cells (lanes 2 and 4). Loading control, fibronectin. (Bottom) Quantitation of expression from three independent experiments. The expression of collagen polypeptides was normalized to the expression of fibronectin and arbitrarily set as 1 for Vim^+/+ cells. The error bars represent SEM, and statistical significance is indicated. (B) Reduced steady-state levels of collagen α1(I) and α2(I) mRNAs in Vim^−/− cells. (Top) Representative RT-PCR analysis of collagen α1(I), collagen α2(I), and GAPDH mRNA levels in Vim^+/+ (lane 1) and Vim^−/− (lane 2) mouse embryonic fibroblasts. (Bottom) Quantification of mRNA levels in three independent experiments. The expression of collagen mRNAs was normalized to the expression of GAPDH mRNA and arbitrarily set as 1 for Vim^+/+ cells. The error bars represent SEM, and statistical significance is indicated. (C) Decay rates of collagen α1(I) and α2(I) mRNAs in Vim^+/+ and Vim^−/− cells. Transcription was blocked by actinomycin D (ACT-D) in Vim^+/+ and Vim^−/− cells, and collagen α1(I) and α2(I) mRNA levels were measured at 0 h, 6 h, 12 h, and 24 h after the block by RT-PCR. GAPDH mRNA was measured as a loading control. The expression of collagen mRNAs was normalized to the expression of GAPDH mRNA and set as 1 for Vim^+/+ cells. The values for α1(I) mRNA (left) and α2(I) mRNA (right) were plotted for each time point, and error bars representing SEM are shown. (D) Segregation of collagen mRNAs with insoluble vimentin. (Top) Vimentin wt fibroblasts (Vim^+/+; lanes 1 and 2) and vimentin knockout fibroblasts (Vim^−/−; lanes 3 and 4) were fractionated into soluble (lanes 1 and 3) and insoluble (lanes 2 and 4) fractions. The fractions were analyzed for the presence of collagen α1(I), collagen α2(I), and actin mRNAs by RT-PCR. (Bottom) Percentages of collagen mRNAs found in the insoluble fractions of Vim^+/+ and Vim^−/− cells, as estimated by three independent experiments. Statistical significance and error bars representing SEM are shown.