Volume 52, Issue 4, Pages 1213-1222 (October 2007) Cadherin-11 is Expressed in Detrusor Smooth Muscle Cells and Myofibroblasts of Normal Human Bladder  Kamiel A.J. Kuijpers, John P.F.A. Heesakkers, Cornelius F.J. Jansen, Jack A. Schalken  European Urology  Volume 52, Issue 4, Pages 1213-1222 (October 2007) DOI: 10.1016/j.eururo.2007.01.052 Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 1 Expression of adherens junction molecules and cytoskeletal proteins in the urothelial and suburothelial layer. Specimen overview by haematoxylin-eosin stain showing multiple layers of flattened cells just below the urothelial lining (A). Note urothelium and underlying layer of flat myofibroblasts (arrows A). Note autofluorescent collagen fibres in negative control (insert B). Clear punctate expression of cadherin-11 (B), α-catenin (C), and β-catenin (D) was found in the flattened suburothelial cells. These cells also expressed smooth muscle actin (E), vimentin (F), and connexin-43 (data not shown). Arrow heads (C and D) show linear, nonpunctate structures, most probably embodying small neurones and vessels. Binocular epifluorescent microscopy, magnification ×100, 4μm. European Urology 2007 52, 1213-1222DOI: (10.1016/j.eururo.2007.01.052) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 2 Costaining of cadherin-11 (green), smooth muscle actin (red), and nuclei (blue) in the urothelium and suburothelial layer. Arrow heads mark thin urothelial layer. Suburothelial myofibroblasts revealed punctate staining for cadherin-11. Punctate labelling disappeared at the layer of collagenous tissue, which was detected as green fibres due to autofluorescence (arrow bottom left). Insert shows enlargement of a few suburothelial myofibroblasts. Erratic expression of actin filaments seemed to associate with membrane-located cadherin-11. Note the nucleus staining centrally in the cell. Binocular epifluorescent microscopy, magnification ×400, 4μm. European Urology 2007 52, 1213-1222DOI: (10.1016/j.eururo.2007.01.052) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 3 Expression of adherens junction molecules and cytoskeletal proteins in detrusor smooth muscle cells. Specimen overview by haematoxylin-eosin stain showing no loosened fascicular structure or widening of intercellular spaces between individual smooth muscle cells (A). No immunoreactions were detected in negative controls (insert B). Detrusor smooth muscle cells expressed clear punctate staining of cadherin-11 (B) and β-catenin (D). “In-line” expression reveals the spindle-like shape of smooth muscle cells. No expression of α-catenin was found (C). Smooth muscle cells expressed smoothelin (E) and smooth muscle actin (F). Binocular epifluorescent microscopy, magnification ×100, 4μm. European Urology 2007 52, 1213-1222DOI: (10.1016/j.eururo.2007.01.052) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 4 Costaining for smooth muscle actin with phalloidin (red), cadherin-11 (green), and nuclei (blue) in transverse sections (A) of detrusor smooth muscle bundles. Cadherin-11 expression seemed localised at the edges of cytoplasmatic phalloidin-positive protrusions, leaving the flat sides in between unoccupied. However, “in-line” expression of cadherin-11, as found using a longitudinal approach (B), showed a punctate pattern that occupied virtually the entire cell membrane. Like in the suburothelial myofibroblasts, cadherin-11 and actin stainings were closely associated. Binocular epifluorescent microscopy, magnification ×400, 4μm. European Urology 2007 52, 1213-1222DOI: (10.1016/j.eururo.2007.01.052) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 5 Confocal image showing double labelling of cadherin-11 (green) and propidiumjodide (red) for nuclei in a few detrusor smooth muscle cells. Cadherin-11 protein was expressed as large dots. Note lack of expression of cadherin-11 within smooth muscle cell cytoplasm and nucleus. Cadherin-11 signal seemed membranously located, describing an “in-line” configuration. Neighbouring cells lack nucleus staining, most likely because of decentral transection of these cells. Confocal epifluorescent microscopy, magnification ×10,000. European Urology 2007 52, 1213-1222DOI: (10.1016/j.eururo.2007.01.052) Copyright © 2007 European Association of Urology Terms and Conditions