Histological phenotypes in the smooth muscle specific cFN iKO mice.

<p>Control mice and cFN iKO mice were treated with tamoxifen from P1 to P3. <b>(A and B)</b> Masson’s trichrome stained cross sections of descending aorta at 3 months of age demonstrate higher amounts of collagen deposition in the media and loose collagen deposition in adventitia of cFN iKO mice (turquoise stain) (B) as compared to controls (A) (<i>n</i> = 3). <b>(C and D)</b> Quantification of collagen in the tunica media (C) and the tunica adventitia <b>(D)</b> of picrosirius red stained aorta sections from 3-month-old cFN iKO and control mice (<i>n</i> = 4–8). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004812#pbio.2004812.s006" target="_blank">S1 Data</a>. <b>(E and F)</b> Masson’s trichrome stained cross sections of descending aorta demonstrate dense collagen deposition in the adventitia of cFN iKO mice at 8 months <b>(F; triangles)</b> as compared to controls <b>(E; triangles)</b> (<i>n</i> = 3). M and A indicate tunica media and tunica adventitia, respectively, and the scale bar represents 50 μm in A–B and E–F. <b>(G–H)</b> Quantification of collagen in the tunica media <b>(G)</b> and tunica adventitia <b>(H)</b> of picrosirius red stained sections of aorta of 8-month-old cFN iKO and control mice (n = 4–8). Parameters quantified are total collagen intensity (Total), mean collagen pixel intensity (Mean), and area of collagen distribution in μm² (Area) in the tunica media/adventitia. Note that the changes in collagen deposition observed at 3 months <b>(A–D)</b> revert back at 8 months <b>(E–H)</b>. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004812#pbio.2004812.s006" target="_blank">S1 Data</a>. <b>(I and J)</b> Immunofluorescence staining of the aortic wall with anti-EDA FN antibody (specific for cFN) shows an effective deletion and reduction of cFN in cFN iKO mice <b>(I)</b> as compared to control mice <b>(J)</b> (arrowheads) (<i>n</i> = 3). <b>(K)</b> Quantification of immunofluorescence staining shown in <b>(I)</b> and <b>(J)</b>. <b>(L and M)</b> Immunofluorescence staining of aortae from control and cFN iKO mice with an antibody against total FN (pFN and cFN). Note there is no reduction of total FN staining in the media of cFN iKO mice <b>(L)</b> as compared to the control mice <b>(M)</b> (<i>n</i> = 4). <b>(N)</b> Quantification of immunofluorescence staining shown in <b>(L)</b> and <b>(M)</b>. Underlying data for (K) and (N) are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004812#pbio.2004812.s006" target="_blank">S1 Data</a>. Scale represents 50 μm in Fig 2I, J, L, M. Lumen is indicated with an asterisk in A–B, E–F, I–J, and L–M. For all statistical analyses, * denotes <i>p</i> ≤ 0.05, ** denotes <i>p</i> ≤ 0.01, and *** denotes <i>p</i> ≤ 0.001. cFN, cellular fibronectin; cFN iKO, cellular fibronectin inducible knockout; Cntrl, control–(<i>Fn(fl/fl</i>); EDA, extra domain-A; FN, fibronectin; ns, denotes not significant; pFN, plasma fibronectin.