Caveolin-1, a Novel Ku70-Interacting Protein Figure 5. Knockdown of caveolin-1 protein expression enhances the dissociation of Bax from Ku70 and the activation of Bax induced by chemotherapeutic drugs. (A, C, D and E) 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 either 5-FU (A, C and D) or etoposide (A and E) for 24 hours. In (B), untransfected cells were treated with 5-FU for 24 hours. In (A), cell lysates were immunoprecipitated with an antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-Ku70 and anti-Bax IgGs. In (B), cell lysates were immunoprecipitated with either anti-caveolin-1 IgGs or anti-Bax IgGs. Immunoprecipitates were then subjected to immunoblotting analysis with antibody probes specific for caveolin-1, Ku70 and Bax. In (C and D), cell lysates were immunoprecipitated with a Bax (6A7) antibody and immunoprecipitates subjected to immunoblotting analysis with anti-Bax IgGs. A blot that is representative of three independent experiments is shown in (C), quantification of Bax levels pulled down by the Bax (6A7) antibody is shown in (D). Values in (D) represent mean 6 SEM; *P,0.001. In (E), mitochondrial fractions were isolated and expression of Bax in mitochondria was detected by immunoblotting analysis using an antibody probe specific for Bax. doi:10.1371/journal.pone.0039379.g005 expressing HCT116 Bax cells, as compared to scrambled siRNA-expressing HCT116 Bax cells (Figure 6B), suggests that, in addition to Bax-dependent apoptosis, caveolin-1 may also inhibit, to a certain extent, Bax-independent cell death following treatment with 5-FU. The Interaction of Ku70 with Caveolin-1 is Required for the Ku70-dependent Protection against Chemotherapeutic Drug-induced Apoptosis Overexpression of Ku70 has been shown to protect against apoptosis [22,25]. Based on our data showing that binding of caveolin-1 to Ku70 protects the cells against apoptosis induced by chemotherapeutic drugs, we would predict less protection against apoptosis by a mutant form of Ku70 that cannot bind to caveolin- 1. Since we show in Figure 3 that caveolin-1 interacts with Ku70, to directly test our hypothesis, we generated a mutant form of Ku70 in which the critical aromatic residues of the caveolin binding domain of Ku70 were mutated to alanines (WRA-Ku70). We show that the in vitro binding of WRA-Ku70 to caveolin-1 was greatly inhibited, as compared to wild type Ku70 (Figures S5B and S5C). We then overexpressed wild type Ku70 and WRA-Ku70 in HCT116 cells. Cells were treated with 5-FU and the interaction of caveolin-1 with Ku70 and Bax was examined by coimmunopre- cipitation studies. We find that 5-FU promoted the interaction of caveolin-1 with wild type Ku70 and Bax in cells overexpressing wild type Ku70 (Figure 7A). In contrast, the interaction of caveolin-1 with WRA-Ku70 and Bax was only marginally stimulated by 5-FU treatment in cells overexpressing WRA- Ku70 (Figure 7A). Consistent with our findings, WRA-Ku70, similarly to wild type Ku70 (Figure 4B), did not co-localize with caveolin-1 in HCT116 cells under resting conditions, being mainly localized in the nucleus and, to a lesser extent, diffuse in the cytoplasm (see arrowheads in Figure S6). However, in contrast to wild type Ku70 (Figure 4B), 5-FU failed to promote the co- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39379