Figure 4.

Loss of Cav-1 and oxidative stress both induce DNA damage in fibroblasts. (A) BSO promotes Cav-1 downregulation and DNA double strand breaks. To induce oxidative stress, hTERT-fibroblasts were grown in 10% NuSerum and treated with increasing concentrations of the glutathione synthase inhibitor BSO or with vehicle alone (H₂O) for 24–48 hours. Then, cells were subjected to western blot analysis with antibodies against Cav-1 and gamma-H2AX. β-actin was used as an equal loading control, and remained unchanged after 24 and 48 hours of BSO treatment. Note that BSO treatment downregulates Cav-1 expression levels and promotes DNA double strand breaks in a concentration-dependent fashion. Consistent with the idea that Cav-1 has a very long half-life, Cav-1 downregulation is detected only after 48 hours of BSO treatment. Conversely, gamma-H2AX levels are increased already after 24 hours of BSO treatment. (B) Cav-1 knock-down promotes DNA double strand breaks. hTERT-fibroblasts treated with Cav-1 siRNA or control siRNA were subjected to Western blot analysis using antibodies against Cav-1 and gamma-H2AX. β-actin was used as an equal loading control. Note that siRNA-mediated Cav-1 downregulation is sufficient to greatly induce DNA double strand breaks.