and with the same concentration of 5-FU (25 mg/ml) for different periods of time (12 h, 24 h and 48 h) (Figures S1A and S1B). Cells were then stained with DAPI and apoptosis was quantified by counting the number of cells showing nuclear condensation. We show that 5-FU induced apoptosis in HCT116 cells transfected with scrambled siRNA and that 5-FU-induced apoptosis was significantly enhanced in HCT116 cells in which caveolin-1 protein expression was reduced by siRNA. These data were confirmed by annexin V staining followed by flow cytometry analysis (Figure 1C) and immunoblotting analysis using antibody probes specific for cleaved caspase 3 and Poly (ADP-ribose) polymerase (PARP) (Figure 1D). Studies have implicated reactive oxygen species (ROS) in 5-FU-mediated apoptosis in colon cancer cells [28]. To independently support our findings, we subjected HCT116 cells transfected with either scrambled or caveolin-1 siRNA to oxidative stress and apoptosis was quantified by DAPI staining. We found that knockdown of caveolin-1 protein expression potentiated oxidative stress-induced apoptosis in HCT116 cells (data not shown). Caveolin-1, a Novel Ku70-Interacting Protein Long-term clonogenic assays provide a more stringent mea- surement of cell survival than short-term assays. To determine the effect of reduced caveolin-1 expression on the long-term survival of colon cancer cells, HCT116 cells were transfected with either scrambled or caveolin-1 siRNA, treated with different concentra- tions of 5-FU (3.125 mg/ml, 6.25 mg/ml, 12.5 mg/ml and 25 mg/ ml) and subjected to crystal violet staining 7 days after 5-FU treatment. Figures 2A and 2B show that knockdown of caveolin-1 protein expression reduced the long-term survival of HCT116 cells, even at the lowest concentration of 5-FU (3.125 mg/ml). Interestingly, the number of clones, after crystal violet staining, was reduced by the knockdown of caveolin-1 protein expression in HCT116 cells even before drug treatment (Figure 2A). Since caveolin-1 siRNA also reduced the number of cells in the S phase of the cell cycle by 13%, without promoting apoptosis, before treatment with 5-FU (Figure S2), caveolin-1 appears to have a positive effect on cell growth in HCT116 cells in addition to its anti-apoptotic function after stimulation with chemotherapeutic drugs. This is consistent with data showing pro-tumorigenic properties of caveolin-1 in colon cancer, as demonstrated by the Figure 1. Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis. HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (A) Endogenous caveolin-1 expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. (B–D) One day after transfection, HCT116 cells were treated with different concentrations of 5-FU for 48 hours. Untreated cells were used as control. In (B), 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 (C), cells were subjected to Annexin V staining and flow cytometry. Values represent mean. In (D), 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.g001 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39379