Telomerase (TRAP-)assay The TRAPEZE® Gel-Based Telomerase Detection assay (Chemicon International, Temecula, CA, USA) was performed according to the manufacturer’s protocol using the isotopic detection. HBCEC populations from two different buy EVP4593 patients were tested, whereby one was obtained after 308d of tumor tissue culture. HBCEC from the other patient were collected after 152d of tumor tissue culture both, by trysinization or by scraping with a rubber policeman. The human embryonic kidney (HEK) cell line 293T was obtained by trypsinization of a steady state culture and used as a positive

control. Briefly, HBCEC and 293T control cells were washed with ice-cold PBS and homogenized in 100 μl ice-cold 1× CHAPS lysis buffer (Chemicon). After incubation for 30 min on ice, the homogenates were centrifuged (12000 g/30 min/4°C) and the supernatants were transferred to a new tube and subjected to a protein quantification measurement using the BCA protein assay. According to the Chemicon protocol,

the TS primer were radioactively end-labeled with γ-32P-ATP before the telomeric repeat amplification reaction was set up to allow the isotopic detection (see Chemicon protocol). Each assay included an internal standard (36 bp band) to control the amplification efficiency. A primer-dimer and PCR contamination control was performed by substituting the selleck cell extract with 1× CHAPS lysis buffer. For data analysis, 25 μl of the amplified product were loaded on a 12.5% non-denaturating PAGE in 0.5× TBE buffer and eventually visualized using a PhosphorImager (GE Healthcare, Freiburg, Germany). ATP release assay following treatment with chemotherapeutic

compounds The effects of chemotherapeutic reagents on two different primary HBCEC were analyzed using the luciferin-luciferase-based ATP tumor chemosensitivity assay (ATP-TCA). Cytotoxicity was determined by measuring the luminescence of luciferin that is proportional to the ATP-release of intact cells. Triplicates of about 1.5 × 104 HBCEC were incubated with different concentrations of chemotherapeutic compounds (Taxol (Bristol-Myers-Squibb); Epothilone A and B (kind gift from Prof. G. Höfle, Helmholtz Center for Infection Research, Braunschweig, Germany); Epirubicin (Pharmacia&Upjohn); Doxorubicin (Sigma)) in a 96-well plate for 6d at 37°C, 5% CO2. The ATP-TCA PtdIns(3,4)P2 assay was performed according to the manufacturer’s protocol (DCS Diagnostica GmbH, Hamburg, Germany) using non-treated cells and cells incubated with the Maximum ATP-inhibitor Solution (DCS) as controls together with an ATP standard. Following lysis of the tumor cells with an selleck kinase inhibitor extraction buffer (DCS), the luminescence was measured in a fluoro/luminometer (Fluoroskan Ascent FL Labsystems, Thermo Scientific, Dreieich, Germany) after addition of the luciferin-luciferase reagent and the percentage of intact (viable) cells was calculated using the Ascent software (Thermo Scientific).