Protein Extraction and Immunoblotting Cells were treated as indicated and exactly harvested by centrifugation at 300 g for 30 seconds. Following resuspension in 1 ml of ice-cold PBS and transfer to 1.5-ml microfuge tubes, cells were spun at 13200 rpm for 30 seconds. The pellet was lysed by incubation for 30 minutes in 200 ��l of cold cell lysis buffer containing 50 mM Tris-HCl (pH:8.0), 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, protease and phosphatase inhibitor cocktails, and Nonidet P-40 1% (v/v). After centrifugation at 13200 rpm for 10 minutes, supernatant containing the total protein extract was removed and stored at ?80��C. Protein concentrations were determined by DC protein assay (Bio-Rad, Munich, Germany).

Proteins (30 ��g) were mixed with loading buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0,004% bromophenol blue, 0,125 M Tris-HCl pH:6,8) and separated on 10�C15% SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 5% blocking reagent (ECL Advance, Amersham Pharmacia Biotech, Freiburg, Germany) in PBS-Tween20 and incubated with appropriate primary and HRP-conjugated secondary antibodies (Amersham Pharmacia Biotech, Freiburg, Germany) in 5% blocking reagent. After required washes with PBS-Tween20, proteins were finally analyzed using an enhanced chemiluminescence detection system (ECL Advance, Amersham Pharmacia Biotech, Freiburg, Germany) and exposed to Hyperfilm-ECL (Amersham Pharmacia Biotech, Freiburg, Germany). Transfections HCT116 wt cells were transfected with Bcl-2 and Bim expression plasmids (Addgene Inc.

, Cambridge, MA, USA) using FuGene 6 transfection reagent (Roche, F. Hoffman-La Roche Ltd., Basel, Switzerland) according to the manufacturer��s protocol. Total protein extraction was performed after 24 hours following completion of transfection procedure for confirmation of ectopic expression. Plasmid-transfected cells were treated with PMC-A as indicated and analyzed by flow cytometry after 24 hours. Statistical Analysis All the illustrated results represent one of at least three independent experiments with similar outcomes. All numerical data are presented as as means �� SD. Statistical significance of responsive differences among differentially treated or differentially transfected cell populations were assessed with unpaired or paired student��s t-test, respectively. Values of P<0.05 and P<0.

01 are marked as * or **, respectively. Results PMC-A Proves More Cytotoxic Compared to Its Precursor PMC and Several Analogs against HCT116 Cells PMC was previously shown to selectively damage vascular endothelial cells in in vitro functional studies on rat aorta and dog arteries [1], [21], [22]. PMC Cilengitide and analogs were also demonstrated to cause cell death in cultured bovine vascular endothelial and Jurkat T leukemia cells [1], [2].