L cells. Moreover, there was no evidence of any inflammatory cellular infiltrate present within the urothelium or the underlying bladder mucosa and the expression of the differentiation-associated plaque protein uroplakin 3a, which is important for limiting transcellular permeability across the urothelium [20], was also normal in KK7 Knockout MiceK7 Knockout MiceFigure 3. Histological Autophagy analysis of K7 knockout tissues. Haematoxylin and eosin stained formalin-fixed tissue sections from wildtype (A, C, E, G) and inhibitor homozygous K7 knockout mice (B, D, F, H). Images show the cortical collecting tubules of kidney (A, B), bile ducts in liver (C, D), pancreatic ducts (E, F) and columnar epithelium of uterus (G, H). Scale bars = 50 mm. doi:10.1371/journal.pone.0064404.gknockout mice suggesting that the urothelial barrier was intact. Further study is therefore required in order to understand how the loss of K7 leads to changes in urothelial cell proliferation. Our analysis of the fate of remaining simple keratins in K7 knockout mice suggests that K7 is required in part for the stabilisation of K18 in vivo. Unlike K18 knockout mice which showed complete loss of K7 [11], in K7 knockout mice Kprotein levels were only reduced but this appears to be a tissuedependent effect since it was only noted in the bladder and in the kidney. In contrast 16985061 to K18, the tail-less type I keratin K19, despite extensive co-expression with K7 in all of the tissues we examined, did not appear to be affected by the loss of K7 suggesting that K19 must be stabilised through pairing with another type II keratin, most likely K8. Although we only found minimal overlap betweenFigure 4. Loss of K7 is associated with hyperproliferation but not hyperplasia of the bladder urothelium. 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 23148522 in the bladder of homozygous K7 knockout mice (arrowheads in F). Scale bars = 50 mm. 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 +/ 2300) urothelial cell nuclei were counted. Standard errors (SE) are indicated by the capped lines. * indicates a p value of less than 0.05 (WT p = 0.01; HET p = 0.007). H. H E stained sections of the bladder urothelium of wildtype, heterozygous and homozygous K7 knockout mice. Scale bar = 25 mm. doi:10.1371/journal.pone.0064404.gK7 Knockout MiceFigure 5. Simple keratin expression in the bladder of K7 knockout mice. A. Coomassie blue stained SDS-PAGE gel and western blots of cytoskeletal-enriched extracts prepared from the bladder, lung and colon of wildtype (+/+), heterozygous (+/2) and homozygous (? K7 knockout mice probed with antibodies to K8, K18, K19 and K20. A monoclonal antibody to b-actin was used to monitor protein loading and was co-incubated with the polyclonal K7 antibody on the same blot. M denotes molecular weight standards, sizes in kDa are as indicated. B. Quantitative RT-PCR ofK7 Knockout Micebladder cDNA from wildtype, heterozygous and homozygous K7 k.L cells. Moreover, there was no evidence of any inflammatory cellular infiltrate present within the urothelium or the underlying bladder mucosa and the expression of the differentiation-associated plaque protein uroplakin 3a, which is important for limiting transcellular permeability across the urothelium [20], was also normal in KK7 Knockout MiceK7 Knockout MiceFigure 3. Histological analysis of K7 knockout tissues. Haematoxylin and eosin stained formalin-fixed tissue sections from wildtype (A, C, E, G) and homozygous K7 knockout mice (B, D, F, H). Images show the cortical collecting tubules of kidney (A, B), bile ducts in liver (C, D), pancreatic ducts (E, F) and columnar epithelium of uterus (G, H). Scale bars = 50 mm. doi:10.1371/journal.pone.0064404.gknockout mice suggesting that the urothelial barrier was intact. Further study is therefore required in order to understand how the loss of K7 leads to changes in urothelial cell proliferation. Our analysis of the fate of remaining simple keratins in K7 knockout mice suggests that K7 is required in part for the stabilisation of K18 in vivo. Unlike K18 knockout mice which showed complete loss of K7 [11], in K7 knockout mice Kprotein levels were only reduced but this appears to be a tissuedependent effect since it was only noted in the bladder and in the kidney. In contrast 16985061 to K18, the tail-less type I keratin K19, despite extensive co-expression with K7 in all of the tissues we examined, did not appear to be affected by the loss of K7 suggesting that K19 must be stabilised through pairing with another type II keratin, most likely K8. Although we only found minimal overlap betweenFigure 4. Loss of K7 is associated with hyperproliferation but not hyperplasia of the bladder urothelium. 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 23148522 in the bladder of homozygous K7 knockout mice (arrowheads in F). Scale bars = 50 mm. 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 +/ 2300) urothelial cell nuclei were counted. Standard errors (SE) are indicated by the capped lines. * indicates a p value of less than 0.05 (WT p = 0.01; HET p = 0.007). H. H E stained sections of the bladder urothelium of wildtype, heterozygous and homozygous K7 knockout mice. Scale bar = 25 mm. doi:10.1371/journal.pone.0064404.gK7 Knockout MiceFigure 5. Simple keratin expression in the bladder of K7 knockout mice. A. Coomassie blue stained SDS-PAGE gel and western blots of cytoskeletal-enriched extracts prepared from the bladder, lung and colon of wildtype (+/+), heterozygous (+/2) and homozygous (? K7 knockout mice probed with antibodies to K8, K18, K19 and K20. A monoclonal antibody to b-actin was used to monitor protein loading and was co-incubated with the polyclonal K7 antibody on the same blot. M denotes molecular weight standards, sizes in kDa are as indicated. B. Quantitative RT-PCR ofK7 Knockout Micebladder cDNA from wildtype, heterozygous and homozygous K7 k.
