Ku70) fails to inhibit chemotherapeutic drug-induced apoptosis. Thus, the interaction of Ku70 with caveolin-1 is critical for the ability of Ku70 to inhibit apotosis. We also found that overexpression of WRA Ku70, in contrast to wild type Ku70, fails to inhibit the Bax/Ku70 dissociation after stimulation with chemotherapeutic drugs, which explains, at the molecular level, why WRA Ku70 fails to protect against chemotherapeutic drug- induced apoptosis. Thus, under cellular conditions where Ku70 fails to interact with caveolin-1 (i.e. cells in which caveolin-1 protein expression is knockdown by siRNA or cells expressing the WRA mutant form of Ku70 that does not interact with caveolin- 1), the ability of Ku70 to limit the release of Bax is prevented. Together, our data indicate that disruption of the interaction between caveolin-1 and Ku70 enhances apoptosis and propose the caveolin-1/Ku70 complex as a novel potential therapeutic target for the treatment of colon cancer. Supporting Information Figure S1 Quantification of nuclear condensation after knockdown of caveolin-1 protein expression in 5-FU- treated HCT116 colon cancer cells. HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, cells were treated with 5-FU for different periods of time. Untreated cells were used as control. Cells were then stained with DAPI. Representative images are shown in (A). Arrows show examples of cells with nuclear condensation. The number of cells showing nuclear condensation was quantified in (B). Values represent mean 6 SEM; *P,0.001. (TIF) Figure S2 Knockdown of caveolin-1 protein expression reduces the number of cells in the S phase of the cell cycle. HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, HCT116 cells were collected, incubated with propidium iodide and subjected to FACS analysis with the use of a fluorescence-activated cell sorter (FACStar plus; Becton Dickinson). A representative cell cycle analysis experiment is shown. Values represent mean from independent experiments. (TIF) Figure S3 Quantification of nuclear condensation after knockdown of caveolin-1 protein expression in 5-FU- treated HT29 colon cancer cells. HT29 colon cancer cells were transfected with siRNA directed against caveolin-1. Trans- fection with scrambled siRNA was used as control. One day after transfection, cells were treated with 5-FU for different periods of time. Untreated cells were used as control. Cells were then stained with DAPI. Representative images are shown in (A). Arrows show examples of cells with nuclear condensation. The number of cells References 1. Palade GE (1953) The fine structure of blood capillaries. J Appl Physics 24: 1424. 2. Yamada E (1955) The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1: 445–458. 3. Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, et al. (1997) Cell- type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 272: 29337– 29346. 4. Matveev S, Li X, Everson W, Smart EJ (2001) The role of caveolae and caveolin in vesicle-dependent and vesicle-independent trafficking. Adv Drug Deliv Rev 49: 237–250. 5. Galbiati F, Razani B, Lisanti MP (2001) Emerging themes in lipid rafts and caveolae. Cell 106: 403–411. Caveolin-1, a Novel Ku70-Interacting Protein showing nuclear condensation was quantified in (B). Values represent mean 6 SEM; *P,0.001. (TIF) Figure S4 Knockdown of caveolin-1 protein expression inhibits long-term survival of HCT116 cells after treatment with etoposide. HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, HCT116 cells were treated with different concentra- tions of etoposide for 1 hour. Untreated cells were used as control. Cells were then cultured for 7 days and stained with crystal violet. A representative crystal violet staining after etoposide treatment is shown in (A). Quantification of crystal violet staining after etoposide treatment is shown in (B). Values represent mean 6 SEM; *P,0.001; #P,0.005. (TIF) Figure S5 5-FU, but not etoposide, increases caveolin-1 mRNA levels in HCT116 cells. The binding of W RA Ku70 to caveolin-1 is dramatically compromised. (A) HCT116 cells were treated with either 5-FU or etoposide for 24 hours. Untreated cells were used as control. RNA was extracted and RT-PCR was performed using primers specific for human caveolin-1. Amplification of GAPDH was performed by RT-PCR as an internal control. A representative blot is shown. (B) Ponceau S staining of GST alone and the GST-caveolin-1 (82–101) fusion protein. (C) GST-caveolin-1 fusion protein pull-down assays were performed using cell lysates from HCT116 cells transiently transfected with either wild type Ku70-HA or WRA Ku70-HA. A blot that is representative of two independent experiments is shown. (TIF) Figure S6 WRA Ku70 fails to co-localize with caveolin-1 after treatment with 5-FU. HCT116 colon cancer cells were transfected with HA-tagged WRA Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as control. Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei were detected by DAPI staining (Blue). Representative images are shown. (TIF) Acknowledgments We thank Dr. Bert Vogelstein for providing HCT116 and HCT116 Bax2/2 cells. Author Contributions Conceived and designed the experiments: HZ DV FG. 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PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39379