Increased acetylation of Ku70 disrupts the Ku70-Bax interaction and inhibits the antiapoptotic function of Ku70 [25]. Finally, peptides based on the amino acid sequence of the Bax-binding domain of Ku70 bind Bax and inhibit cell death [26,27]. Thus, by interacting with Ku70, Bax is sequestered away from mitochon- dria and is maintained in an inactive state. The molecular mechanism underlying the sequestration of Bax by Ku70 and the release of Bax from Ku70 after stimulation with an apoptotic inducer remains to be fully identified. In this study, we focused on the functional role of caveolin-1 in chemotherapeutic-induced apoptosis of colon cancer cells and the underlying molecular mechanism. We found that Ku70 is a novel caveolin-1-binding protein and that binding of caveolin-1 to Ku70 inhibits chemotherapeutic drug-induced apoptosis of colon cancer cells by limiting the release of Bax from Ku70. Materials and Methods Materials Antibodies and their sources were as follows: anti-caveolin-1 (2297) and anti-Bax (6A7) IgGs were from BD Transduction Laboratories (Lexington, KY); anti-caveolin-1 (H-97), anti-Ku70 (A-9) and anti-Bax (N-20) IgGs were from Santa Cruz Biotech- nology (Santa Cruz, CA); anti-cleaved caspase-3 and anti-cleaved PARP IgGs were from Cell Signaling Technology (Danvers, MA); horseradish peroxidase-conjugated goat anti-mouse and anti- rabbit secondary antibodies were from Pierce (Rockford, IL, USA). McCoy’s 5a medium and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA). Other biochemicals used were of the highest purity available and were obtained from regular commercial sources. Cell Culture and Drug Treatment Human colon carcinoma cells HT29, HCT116 Bax +/2 (HCT116) and HCT116 Bax 2/2 (HCT116 Bax null) (a generous gift from Dr. Bert Vogelstein) were grown in McCoy’s 5a medium containing glutamine, antibiotics and 10% FBS. Cells were incubated in growth medium supplemented with 50, 25, 12.5, 6.25, or 3.125 mg/ml of 5-FU for 12, 24 or 48 hours. HCT116 and HCT116 Bax cells were also incubated in growth medium supplemented with 800, 600, 400 or 200 mM of etoposide for 24 hours. siRNA Treatment Knockdown of caveolin-1 protein expression was achieved by transfection of HCT116 and HT29 cells with small interfering RNA (siRNA) duplexes. The target sequence was as follows: 59- aaccagaagggacacacag-39. Scrambled siRNA was used as a negative control. Transfection was performed using Lipofectamine 2000 according to the manufacture’s recommendations. Cloning of wild type (WT) and WRA Ku70 constructs. The entire coding region of mouse Ku70 was amplified by polymerase chain reaction amplification using a full- length cDNA clone (ID 3669715) from Open Biosystems (Huntsville, AL) and the following primers: forward 59-gaattcggt- cagagtgggagtcctactac-39 and reverse 59-ctcgagtcagttcttctccaagtgtct- 39. The amplified DNA was sequenced to rule out the introduction of unwanted mutations. The PCR fragment was digested to create unique EcoRI and XhoI ends and subcloned into the pCMV-HA expression vector (WT Ku70-HA). The WRA mutant of Ku70 (WRA Ku70-HA) was generated by replacement of the aromatic residues within the caveolin binding motif of Ku70 (amino acids 471–478) with alanines by polymerase chain reaction amplifica- tion using appropriate internal primers (forward: 59- Caveolin-1, a Novel Ku70-Interacting Protein gttcaaaagctccgcgccacagccagatctgacagtgctgagaatccagtcctg-39; re- verse: 59-caggactggattctcagcactgtcagatctggctgtggcgcggagcttttgaac- 39) and subcloned EcoRI and XhoI into the pCMV-HA expression vector. Transfection of WT Ku70-HA and WRA Ku70-HA in HCT116 cells was performed using Lipofectamine 2000 according to the manufacture’s recommendations. Western Blotting Assay Protein samples were collected in boiling Laemmli buffer (0.2 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol), vigorously resus- pended and boiled for 5 min. Protein concentration was calculated using the BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL). Equal amount of proteins were separated on 12.5% Tris-HCl polyacrylamide gels after the addition of 0.01% bromophenol blue and 20 mM DTT. Protein samples were then transferred onto nitrocellulose membranes overnight at 4uC. Ponceau S staining was performed to confirm equal loading. Blots were incubated for 1 hr in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.2% Tween 20) containing 2% dry milk and 1% bovine serum albumin. After three washes with TBST, membranes were incubated overnight with the primary antibody and for 1 hr with horseradish peroxidase-conjugated secondary IgG. Bound antibodies were detected using a Super Signal chemiluminescent solution (Pierce Chemical, Rockford, IL). DAPI (4,6-Diamidino-2-Phenylindole) Staining HCT116 and HT29 cells (untreated or treated with chemo- therapeutic drugs) were harvested and incubated with staining buffer (PBS containing 3.7% paraformaldehyde, 0.1% Triton, 10 mg/ml RNase A and 1 mg/ml DAPI) at room temperature for 1 hr. Nuclear morphology was examined under a Olympus Provis fluorescent microscope. A total of 1,200 cells were scored from 4 independent viewing areas from three independent experiments for each experimental point. Annexin V staining and flow cytometry analysis. HCT116 cells were transfected with caveolin-1 siRNA and scrambled siRNA as described above. After 24 hours, cells were treated with 25 mg/ml 5-FU for 48 hours. Untreated cells were used as control. The Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen, San Diego, CA) was used to detect apoptosis by flow cytometry. Cells were harvested (including detached cells), and processed according to the manufacturer’s instructions. Fluorescence-activated cell sorting analysis was carried out using a FACScan flow cytometer (Becton Dickinson, San Diego, CA) and CellQuest software. Clonogenic Assay HCT116 cells were transfected with either caveolin-1 siRNA or scrambled siRNA as described above. After 24 hours, cells were treated with different concentrations of either 5-FU (3.125 mg/ml, 6.25 mg/ml, 12.5 mg/ml and 25 mg/ml) or etoposide (200 mM, 400 mM and 600 mM) for 1 hour. Untreated cells were used as control. 500 cells were then plated into 12-well plates and cultured for 7 days. Cells were stained with crystal violet by incubating the cells with 10% crystal violet in 70% ethanol for 2 minutes followed by extensive washes with PBS. Quantification of crystal violet staining from three independent experiments was performed by counting the number of clones using Image J software analysis. Cell Cycle Analysis by Fluorescence-activated Cell Sorter (FACS) Cells were collected by trypsinization, pelleted, and washed with PBS. Cells were resuspended and fixed overnight with 1 ml of ice- PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39379