Figure 11.

Downregulation of Cdc20 promotes phosphorylation-dependent loss of securin in H460 cells. LiCl and TDZD-8 inhibit cell death induced by either Cdc20 siRNA or etoposide. A, H460 cells were transfected with Cdc20 siRNA and cultured for 1, 2, 3, and 6 days. Transfection with control (Ctl) siRNA served as control. Cells were then collected and cell lysates subjected to immunoblotting analysis with anti-Cdc20 and anti-securin IgGs. Ponceau S staining shows equal total protein loading. B, Cdc20 siRNA was transfected in H460 cells. Transfection with control (Ctl) siRNA was performed as control. After 1 day, cell lysates were incubated with lambda protein phosphatase (λ-PP; 800U) for either 3 or 24 h. Untreated samples (N.T.) served as control. Cell lysates were then subjected to immunoblotting analysis with antibody probes specific for Cdc20 and securin. Ponceau S staining shows equal total protein loading. C, different amounts of Cdc20 siRNA (5, 10, 20, and 40 pmol) were transfected in H460 cells. Control (Ctl) siRNA was transfected as control. After 1 day, cells were collected and expression of Cdc20 and securin was determined by immunoblotting analysis with anti-Cdc20 and anti-securin IgGs. Ponceau S staining shows equal total protein loading. D, H460 cells were transfected with Cdc20 siRNA and cultured for 24 h in the presence of LiCl (1 mM and 10 mM). Transfection with control (Ctl) siRNA and treatment with H₂O served as controls. Cells were then collected and securin expression was detected by immunoblotting analysis with anti-securin IgGs. Ponceau S staining shows equal total protein loading. Quantification of P-securin is shown at the bottom of the blot. E, H460 cells were transfected with Cdc20 siRNA and treated with either LiCl (10 mM) or TDZD-8 (1 μM) for 48 h. Transfection with control (Ctl) siRNA and treatment with DMSO served as controls. Cell survival was then quantified by MTT assays. F, H460 cells were treated with etoposide (100 μM) in the presence of either LiCl (10 mM) or TDZD-8 (1 μM) for 48 h. Treatment with DMSO served as control. Cell survival was then quantified by MTT assays. Values in (E) and (F) represent means ± SEM; statistical comparisons were made using the student’s t test. ∗p < 0.005. DMSO, dimethyl sulfoxide.