Volume 54, Issue 3, Pages 612-620 (September 2008) Transient Receptor Potential Vanilloid Type 2 (TRPV2) Expression in Normal Urothelium and in Urothelial Carcinoma of Human Bladder: Correlation with the Pathologic Stage  Sara Caprodossi, Roberta Lucciarini, Consuelo Amantini, Massimo Nabissi, Giacomo Canesin, Patrizia Ballarini, Adriana Di Spilimbergo, Marco Andrea Cardarelli, Lucilla Servi, Gabriele Mammana, Giorgio Santoni  European Urology  Volume 54, Issue 3, Pages 612-620 (September 2008) DOI: 10.1016/j.eururo.2007.10.016 Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 1 TRPV2 expression in human normal urothelial and urothelial carcinoma cell lines. (A) Expression of TRPV2 coding sequence in normal human urothelial cells was evaluated by polymerase chain reaction (PCR) analysis. PCR products were separated by electrophoresis on 1% ethidium bromide-stained agarose gel. Arrows indicate the full-length hTRPV2 (2.3kbp) and the splice-variant s-TRPV2 (1.9kbp). MW=molecular weight. (B) Exon-intron structure of hTRPV2 (NM_016113) and newly reported cDNA s-TRPV2 obtained by the National Center for Biological Information Spidey program. Sequences are aligned with the human chromosome 17 contig (NT_010718). (C) The deduced C-terminal amino acid sequences of s-TRPV2 from normal human urothelial cells (NHUCs) and RT4 cells were compared with human TRPV2 protein. TM=transmembrane domain. (D) cDNA from NHUCs and RT4, EJ, and J82 cell lines was amplified by PCR for TRPV2 expression. PCR products were analysed by electrophoresis on 2% ethidium bromide-stained agarose gel. The hTRPV2-plasmid construct was used as positive control (+). Arrows indicate the two products obtained. MW=molecular weight. European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 2 Expression of TRPV2 protein in human normal urothelial and urothelial carcinoma cell lines. (A) TRPV2 expression was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS) analysis using a goat anti-human TRPV2 polyclonal antibody. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat was used as secondary antibody. The data shown are representative of three separate experiments. White area indicates negative control. Data are expressed as mean fluorescence intensity (MFI). (B) Immunocytochemical localisation of TRPV2 on normal human urothelial cells (NHUCs) and RT4, EJ, and J82 cell lines was evaluated by confocal microscopy. Goat anti-human TRPV2 polyclonal antibody and Texas red-conjugated rabbit anti-goat were used as primary and secondary antibodies, respectively. Bars=10μm. Data shown are representative of three separate experiments. European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 3 TRPV2 expression in normal human bladder. (A) cDNA from a commercial bladder tissue mRNA (HBL) or normal human bladder tissue specimens (NB) was amplified by polymerase chain reaction (PCR) for TRPV2 expression. PCR products were analysed by electrophoresis on 2% ethidium bromide-stained agarose gel. A plasmid containing the human TRPV2 cDNA sequence was used as positive control (+). The NB samples are representative of six specimens analysed. (B) The section shown is a representative field of formalin-fixed, paraffin-embedded NB tissue, immunostained with a goat anti-human TRPV2 polyclonal antibody. Arrows indicate TRPV2+ cells. Magnifications: ×20. L=lumen of the urinary bladder. The NB samples are representative of six specimens analysed. Size bar: 25μm. (C) Lysates from three different NB specimens were separated on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with a goat anti-human TRPV2 polyclonal and mouse anti-human α-tubulin monoclonal antibodies. Sizes are shown in kilodaltons. hTRPV2–plasmid-transfected HeLa cells were used as positive control (+). Arrowheads indicate the bands consistent with the full-length and short-form TRPV2, and with the α-tubulin protein. The NB samples are representative of six specimens analysed. Data are representative of three separate experiments. European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 4 TRPV2 short-form expression in urothelial carcinoma tissues. (A) cDNA from pTa–pT4-staged UC tissues was amplified by polymerase chain reaction (PCR) for TRPV2 expression. PCR products were analysed by electrophoresis on 2% ethidium bromide-stained agarose gel. A plasmid containing the human TRPV2 cDNA sequence was used as positive control (+). For each tissue type two of six analysed specimens are shown. (B) Short-form TRPV2 expression in normal bladder tissues and different stages of urothelial carcinoma specimens. aThe p value was determined by the Pearson χ2 test and reveals a significant correlation between loss of s-TRPV2 expression and pathologic stages. *p<0.01 was determined by comparing NB with pTa, pT1, pT2 stages, and pTa with pT1 and pT2 stages; ns=not significant by comparing pT1 with pT2 stage. European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 5 TRPV2 expression in UC tissues at different stages. (A and B) TRPV2 mRNA levels from specimens (n=58) of urothelial carcinoma tissues at different stages (pTa–pT4) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR) in triplicate. Results (mean±standard deviation) are normalised for β-actin expression. TRPV2 levels were expressed as relative fold with respect to normal bladder (NB) used as control. Data shown are representative of three separate experiments. Statistical analysis was performed comparing (A) pTa, pT1, pT2, pT3, and pT4 stages with NB (*p≤0.001; **p≤0.0005) and (B) G1 with G2 in pTa samples, G2 with G3 in pT1, pT2, and pT3 samples (*p≤0.05). European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions

Fig. 6 Immunohistochemical analysis of TRPV2 protein in urothelial carcinoma tissues. Sections from paraffin-embedded urothelial carcinoma (UC) tissues at different pathologic stages (pTa–pT4) were immunostained with a goat anti-human TRPV2 polyclonal antibody. Images are representative of six slides for each UC stage. Magnification: ×20. (A) Omission of the primary antibody; (B) pTa UC specimen. The labelling intensity of superficial cells was similar to that in normal bladder tissue. (C and D) UC specimens staged as pT1 and pT2, respectively. A more intense labelling was observed, gradually extended to the basal cells. (E and F) pT3 and pT4 specimens, respectively. The labelling intensity is markedly enhanced in all cell layers, especially in pT4 sample. Size bars: 25μm. European Urology 2008 54, 612-620DOI: (10.1016/j.eururo.2007.10.016) Copyright © 2007 European Association of Urology Terms and Conditions