Caveolin-1, a Novel Ku70-Interacting Protein Figure 2. Caveolin-1 promotes long-term survival and inhibits apoptosis of colon cancer cells after treatment with chemotherapeutic drugs. HCT116 (A, B, E and F) and HT29 (C and D) 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 different concentrations of 5-FU for 48 h (A–D) or etoposide for 24 h (E and F). Untreated cells were used as control. In (A and B), cells were cultured for 7 days and stained with crystal violet. A crystal violet staining that is representative of three independent experiments is shown in (A), quantification of the number of clones after crystal violet staining is shown in (B). Values represent mean 6 SEM; *P,0.001; #P,0.005. In (C and E), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001; #P,0.005. In (D and F), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g002 over-expression of caveolin-1 in colon cancer tissues and colon cancer cells [18–20]. To determine whether the caveolin-1-mediated inhibition of apoptosis is not limited to the combination of HCT116 cells and 5- FU, a different colon cancer cell line, HT29, was treated with different concentrations of 5-FU after caveolin-1 protein expres- sion was knockdown by siRNA (data not shown). Apoptosis was then quantified by DAPI staining (Figures 2C, S3A and S3B) and immunoblotting analysis to detect cleaved caspase 3 and PARP (Figure 2D). We found enhanced 5-FU-induced apoptosis when caveolin-1 protein expression was knockdown in HT29 cells. In addition, scrambled and caveolin-1 siRNA-transfected HCT116 cells were treated with a different chemotherapeutic drug, etoposide. We show enhanced etoposide-induced apoptosis in HCT116 cells transfected with caveolin-1 siRNA, as compared to scrambled siRNA-transfected cells, after DAPI staining (Figure 2E) and immunoblotting analysis to detect activated caspase 3 and PARP (Figure 2F). In addition, the long-term survival of HCT116 cells after treatment with etoposide was inhibited by knockdown of caveolin-1 protein expression using siRNA (Figures S4A and S4B). Ku70 is a Novel Caveolin-1-binding Protein Caveolin-1 is known to interact with signaling molecules carrying a caveolin binding domain [14,15]. Ku70 has been shown to regulate apoptosis [22–25]. Interestingly, Ku70 possesses a putative caveolin binding domain between amino acids 471 and 478 (Figure 3A). To investigate the molecular mechanism underlying the caveolin-1-mediated inhibition of apoptosis, we asked whether Ku70 was a novel caveolin-1-interacting protein. To this end, we performed pulldown assays using a series of caveolin-1 deletion mutants fused to GST (Figure 3B). Figures 3C shows that Ku70 is a novel caveolin-1-binding protein and that the caveolin scaffolding domain (CSD) of caveolin-1 (residues 82–101) is sufficient for binding to Ku70. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39379