Fluorescent images of (A) NEB-1 K14wt-GFP and (B) NEB-1 K14R125P-GFP keratinocytes undergoing incremental uniaxial strain.

<p>The average cell strain is depicted in the top left corner of each image. The cytokeratin networks of the NEB-1 K14R125P-GFP cells withstood extreme cellular strains of 133% and there was no evidence of intermediate filament bundle rupture or the development of keratin aggregates (n = 10). Scale bar = 20 µm.</p

Fluorescent images of the K5/K14, F-actin and microtubule networks in NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes fixed at 0% or 133% strain.

<p>Cells were treated with 1 µg/mL nocodazole (Nc) to disturb the F-actin network, or were untreated (control). The F-actin network was visualized with rhodamine-phalloidin (100 nM), and α-tubulin with immunofluorescence. K14-GFP proteins were expressed and visualized by fluorescence microscopy. Scale bar = 20 µm.</p

The green vital inclusion dye FDA and blue vital exclusion dye DAPI were used to test for necrosis after extreme strain in NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP cells.

<p>The keratinocytes were stretched to a specific strain and then returned to the relaxed state for viability staining. Necrosis increased significantly with increasing cell strain (* = p<0.05; ** = p<0.001). No significant difference in viability was found between the NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes after undergoing extreme uniaxial strain of 133% (p>0.1). Error bars are standard error. Scale bar = 20 µm.</p

Effect of osmotic shock on the keratin cytoskeleton in the two cell lines studied.

<p>A. NEB-1 K14wt-GFP (upper panel) and NEB-1 K14R125P-GFP (lower panel). Both cell lines were subjected to hypo-osmotic shock. Clear peripheral aggregates were seen in R125P cells 20 and 30 min after osmotic shock. No filament fragmentation or aggregates were observed for NEB-1 K14wt-GFP cells. Scale bar = 15 µm. B. Insets from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031320#pone-0031320-g003" target="_blank">Figure 3A</a>, 20 min after osmotic shock, showing K14 aggregates in the NEB-1 K14R125P-GFP cells, and reconstituted K14 filaments in the NEB-1 K14wt-GFP cells.</p

The viability of NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes before (0%) and after (133%) extreme uniaxial strain.

<p>The treatments consisted of 1 µg/mL nocodazole (Nc) to disrupt the microtubule network and 0.5 µM latrunculin A (Lat A) to disrupt the F-actin network (n = 6). There was no significant difference in viability between the control cells and cells treated with nocodazole at extreme strain (p>0.1). Cells treated with Lat A exhibited significantly lower necrosis in both cell lines compared to the control (p<0.01).</p

Fluorescent images of (A) WT NEB-1 cells and (B) EBS mutant (R125P) K14 KEB-7 keratinocytes undergoing incremental uniaxial strain.

<p>The average cell strain is depicted in the top (A) or the bottom (B) of each panel. Both cell lines were subjected to stretch, then fixed and permeabilized and stained with the anti-K14 (LL001, Santa Cruz) monoclonal antibody. The cytokeratin networks of the EBS cells withstood extreme cellular strains of 133% and there was no evidence of intermediate filament bundle rupture or the development of keratin aggregates. Arrowheads indicate wavy K14 intermediate filaments. Scale bar = 20 µm.</p

Effect of osmotic shock on the keratin cytoskeleton in the NEB-1 cell line (A) and the EBS derived cell line KEB-7 (B).

<p>Both cell lines were subjected to hypo-osmotic shock, then fixed and permeabilized and stained for K14 intermediate filaments. Clear peripheral aggregates were seen in KEB-7 cells 30 min after osmotic shock. No filament fragmentation or aggregates were observed for NEB-1 cells. Scale bar = 22 µm.</p

Loss of K7 is associated with hyperproliferation but not hyperplasia of the bladder urothelium.

<p>Immunohistochemistry of bladder sections from wildtype (A, D), heterozygous (B, E) and homozygous K7 knockout mice (C, F) stained with a rabbit polyclonal antibody to K7 (A, B, C) and mouse monoclonal antibody MM1 to the cell proliferation marker Ki-67 (D, E, F). Arrowheads and insets in panels D and E indicate Ki-67 positive nuclei in wildtype (D) and heterozygous K7 knockout (E) bladder. More Ki-67 positive cell nuclei can be seen in the bladder of homozygous K7 knockout mice (arrowheads in F). Scale bars = 50 µm. G. Graph showing the percentage of Ki-67 positive urothelial cells in wildtype, heterozygous and homozygous K7 knockout mice (5 bladders per genotype). For each bladder, 10 random images were collected and an average of 1480 (SD +/−300) urothelial cell nuclei were counted. Standard errors (SE) are indicated by the capped lines. * indicates a <i>p</i> value of less than 0.05 (WT <i>p</i> = 0.01; HET <i>p</i> = 0.007). H. H&E stained sections of the bladder urothelium of wildtype, heterozygous and homozygous K7 knockout mice. Scale bar = 25 µm.</p

<i>Krt7</i> gene targeting strategy.

<p>A. Schematic diagram of the mouse <i>Krt7</i> gene and upstream sequences, only the proximal part of the gene encompassing exons 1 to 3 is shown. Filled black boxes denote exons 1, 2, 3. The long and short homology arms of the targeting vector are indicated by filled black rectangles. Restriction enzyme sites are as indicated and the open black boxes denote the locations of the DNA probes that were used for southern blotting at the 5′ and 3′ ends of recombination in targeted ES cells. B. Genotyping of K7 knockout mice, the wildtype allele is 743 bp, the targeted allele is 593 bp. C. RT-PCR analysis of cDNA from the bladder (lanes 1–3), lung (lanes 4–6), colon (lanes 7–9) and kidney (lanes 10–12) amplified with primers to full-length K7 (∼1.5 kb) and GAPDH (509 bp). Lanes 1, 4, 7 and 10 are from wildtype mice; lanes 2, 4, 6 and 8 are from heterozygote K7 knockout mice; lanes 3, 6, 9 and 12 are from homozygous K7 knockout mice. M = DNA size standards. D. Northern blot of bladder RNA from wildtype (+/+), heterozygous (+/−) and homozygous (–) mice detected with RNA probes to K7 and GAPDH. The position of the 18S ribosomal subunit is indicated by a black bar. E. Coomassie blue SDS-PAGE gel and western blot of cytoskeletal extracts prepared from the bladder (lanes 1, 2, 3), lung (lanes 4, 5, 6) and colon (lanes 7, 8, 9) of wildtype (lanes 1, 4, 7), heterozygous (lanes 2, 4, 6) and homozygous (lanes 3, 6, 9) K7 knockout mice probed with a C-terminal K7 antibody. M = molecular weight standards, sizes in kDa are as indicated.</p

Immunofluorescence analysis of simple keratin expression in K7 knockout mice.

<p> = intensity of staining and localization similar to wildtype tissue.</p>*<p>confirmation by western blotting.</p><p>ne. no protein expression.</p>¶<p>glandular cell staining.</p