cold 70% ethanol at 4uC. On the day of analysis, cells were centrifuged at 3000 rpm for 8 min and the supernatant was carefully removed. Subsequently, cells were resuspended in 1.0 ml of PI solution (PBS containing 0.1% glucose, 1.1 mg/ml RNAse, and 0.05 mg/ml propidium iodide) and incubated at room temperature for 30 min. Cell were then subjected to FACS analysis with the use of a flow cytometer (LSR II, BD biosciences). For each analysis 10,000 gated events were collected to permit cell cycle analysis. Data analysis was performed with the use of CellQuest software. A representative cell cycle analysis from three independent experiments is shown. Glutathione S -transferase (GST) Pull-down Assay GST-caveolin-1 (GST-Cav-1) constructs were as follows: GST fused to full-length caveolin-1 (Cav-1 FL), GST fused to residues 82 to 101 of caveolin-1 (GST-Cav-1 (82–101)) and GST fused to residues 1 to 101 of caveolin-1 (GST-Cav-1 (1–101)). GST-Cav-1 fusion protein constructs were transformed into Escherichia coli (BL21 strain; Novagen, Inc.). After induction of expression through addition of 0.5 mM isopropyl-b-D-galactoside (Sigma), GST-Cav-1 proteins were affinity purified on glutathione-agarose beads, using the detergent Sarcosyl for initial solubilization. GST- Cav-1 and GST alone (bound to glutathione-agarose beads) were washed 3 times with TNET buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing protease inhibitors. SDS-PAGE followed by Comassie staining was used to determine the concentration of GST-Cav-1 per 100 ml of packed bead volume. Pre-cleared cell lysates of HCT116 cells over- expressing HA-Ku70 were diluted in buffer A (10 mM Tris, pH 8.0, 0.1% Tween 20) and added to approximately 100 ml of equalized bead volume for overnight incubation at 4uC. After binding, the beads were extensively washed with phosphate- buffered saline (6 times). Finally, the beads were resuspended in 2X sample buffer (0.4 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue) and subjected to SDS-PAGE. Co-Immunoprecipitation Cells were washed twice with PBS and lysed for 45 min at 4uC in an IP lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octyl glucoside and protease inhibitors; Bax immunoprecipitation was performed using 0.1 mM Hepes and 1% CHAPS). Subsequently, lysates were centrifuged at 13,0006g for 10 min at 4uC. The supernatant was pre-incubated with protein A sepharose beads for 1 hr at 4uC. After centrifugation at 10,000 rpm for 30 sec, the supernatant was collected and protein concentration calculated using the BCA protein assay kit (Thermo Fisher Scientific Inc.). Equal amount of total proteins were incubated overnight at 4uC with anti-caveolin-1 or anti-Bax antibodies and protein A sepharose beads. The immunoprecipitates were washed three times with lysis buffer and re-suspended in Laemmli buffer (0.2 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 20 mM DTT). Following vigorous vortexing and boiling, samples were separated on 12.5% SDS-PAGE. Blots were then probed with appropriated antibodies. Immunoprecipitations with protein A sepharose beads were performed as control and did not produce any detectable band by immunoblotting analysis using the appropriate antibody (data not shown). Mitochondria Isolation Cells were washed once in PBS and resuspended in 600 mL of mitochondrial isolation buffer containing 10 mM Hepes (pH 7.4), 250 mM sucrose, 1 mM EDTA and protease inhibitors. Cells were broken in a Dounce homogenizer for 40 times. Nuclei and unbroken cells were removed by centrifugation at 10006g at 4uC Caveolin-1, a Novel Ku70-Interacting Protein for 15 min. Clarified lysates were subjected to centrifugation at 12,0006g at 4uC for 15 min. The mitochondrion-containing pellets were resuspended in 2x SDS sample buffer. Mitochondrial fractions were positive for the mitochondria marker cytochrome oxidase subunit IV and negative for the cytoplasmic marker alpha- tubulin (data not shown). RNA Isolation and RT-PCR Cells were collected and total RNA was isolated using the RNeasy Mini kit from Qiagen (Valencia, CA). Equal amounts of RNA were treated with RNase-free DNase, and subjected to reverse transcrip- tion using the Advantage RT-for-PCR kit from Clontech (Mountain View, CA), according to the manufacturer’s recommendations. PCR was then performed in the exponential linear zone of amplification for each gene studied. The caveolin-1-specific primers used were the following: forward 59-ctacaagcccaacaacaagg-39; reverse 59- cagacagcaagcggtaaaa-39. A sequence corresponding to GAPDH was also amplified as an internal control. Immunofluorescence Microscopy Cells grown on glass coverslips were washed three times with PBS w/Ca++/Mg++ and fixed for 30 min at room temperature with 2% paraformaldehyde in PBS w/Ca++/Mg++. Fixed cells were rinsed with PBS w/Ca++/Mg++and permeabilized with 0.1% Triton X-100, 0.2% bovine serum albumin for 10 min. Then cells were treated with 25 mM NH4Cl in PBS w/Ca++/Mg++ for 10 min at room temperature to quench free aldehyde groups. Cells were treated with RNase (200 mg/ml) for 10 minutes and with DAPI (1 mg/ml) for 20 minutes. Cells were rinsed with PBS w/Ca++/Mg++ and incubated with the primary antibody (diluted in PBS with 0.1% Triton X-100, 0.2% bovine serum albumin) for 2 h at room temperature. After three washes with PBS w/Ca++/ Mg++ (10 min each), cells were incubated with the secondary antibody for 1 h at room temperature: lissamine rhodamine B sulfonyl chloride-conjugated goat anti-rabbit antibody (5 mg/ml) and fluorescein isothiocyanate-conjugated goat anti-mouse anti- body (5 mg/ ml). Finally, cells were washed three times with PBS w/Ca++/Mg++ (10 min each wash) and slides were mounted with slow-Fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR) and observed using a Zeiss Confocal Microscope (LSM 5 Pascal). Statistical Analysis Quantification studies were performed in triplicate and the average 6 standard error of the mean (S.E.M.) is shown. Significance was calculated using the Student’s t-test. Quantifica- tion of active Bax was obtained by scanning images of western blotting analysis from three independent experiments using Image J software. Immunoprecipitation and immunoblotting analysis were performed three independent times and representative blots are shown. Results Caveolin-1 Inhibits Chemotherapeutic Drug-induced Apoptosis and Promotes Long-term Survival of Colon Cancer Cells To directly address the role of caveolin-1 in chemotherapeutic drug-induced apoptosis in colon cancer cells, we knocked-down caveolin-1 protein expression by siRNA in HCT116 colon cancer cells (Figure 1A). Scrambled and caveolin-1 siRNA-transfected HCT116 cells were treated with different concentrations (3.125 mg/ml, 6.25 mg/ml, 12.5 mg/ml and 25 mg/ml) of the chemotherapeutic drug 5-fluoracil (5-FU) for 48 hours (Figure 1B) PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39379