Rabbit polyclonal antibodies against lamin A/C as well as mouse monoclonal anti-galectin-3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-villin antibodies were kindly provided by Dr. Sylvie Robine (Curie Institute, Paris). Mouse anti α- tubulin antibodies and rabbit anti-β-catenin antibodies were purchased from Sigma (Munich, Germany). Alexa488 and Alexa546 secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Hoechst 33342 from Fluka (Ronkonkoma, NY)

was used for nuclei staining. 2.2 Kidney sample preparation, cell culture and Western blotting Renal cancer samples, intermediate tissue sample and normal tissue samples of the same Selleckchem Palbociclib kidney were obtained from nephrectomy surgeries. The intersection zone between tumor and normal tissue was defined as intermediate tissue. The study was positively evaluated by the local ethic commission. The patients gave a written informed consent for this study and were not followed clinically. After nephrectomy the specimens were stored in ice-cold PBS

containing a protease Erlotinib purchase inhibitor cocktail and samples were immediately processed for Western blotting, immunohistochemistry or nuclear matrix isolation. Epithelial kidney cells (RC-124) and cells of clear cell renal cell carcinoma (RCC-FG1) (Cell Lines Service, Germany) were cultivated in McCoy’s 5a medium/10% FCS (PAA, Pasching, Austria). Western blot analysis was performed essentially as described before [13]. Protein concentrations were Farnesyltransferase established by Bradford protein assay (BioRad DC Protein Assay, Munich, Germany). Equal amounts of 60 μg/slot were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% skimmed milk powder in PBS. Following immunostaining, bands were detected and quantified using Gel-Pro Software (Kapelan Bio-Imaging, Leipzig, Germany) and normalized to the sum or to tubulin quantities of the same sample. 2.3 Histochemistry and immunohistochemistry Kidney samples from normal, intermediate and tumor tissue were cut into sections of 5 mm and fixed with either formalin (3.7%) or Carnoy (60% Ethanol, 30% chloroform, 10% acetic

acid) overnight and processed as previously described [13]. Images of the samples were captured using a confocal microscope TCS SP2 AOBS (Leica, Wetzlar, Germany). Image stacks were deconvoluted and 3D reconstructed by using the Volocity software package (Improvision, Coventry, UK). 2.4 Nuclear matrix isolation Immediately following nephrectomy, nuclear matrix of homogenized tissues was isolated essentially according to [14]. All procedures were performed on ice and all buffers were cooled to 4°C. Normal and tumor tissue samples from human kidney were Dounce homogenized in 2 ml of buffer A (0.25 M sucrose, 20 mM Tris-HCl, 3 mM MgCl2, pH 7.85 supplemented with a protease inhibitor cocktail) followed by centrifugation at 1000 × g for 10 min at 4°C.