Figure 6.

Inhibition of the Cdc20-APC/C pathway promotes phosphorylation-dependent degradation of securin in WI-38 human fibroblasts.A, WI-38 cells were transfected with Cdc20 siRNA and cultured for 1, 2, and 3 days. Transfection with control (Ctl) siRNA was used as control. Cells were collected and the expression of Cdc20 and securin was determined by immunoblotting analysis using anti-Cdc20 and anti-securin IgGs. Ponceau S staining shows equal total protein loading. B, WI-38 fibroblasts were treated with Apcin (50 μM) and pro-TAME (40 μM) for 1, 2, 3, and 6 days. Treatment with DMSO served as control. Cells were collected and cell lysates were subjected to immunoblotting analysis using an antibody probe specific for securin. Ponceau S staining shows equal total protein loading. C, Cdc20 siRNA was transfected in WI-38 fibroblasts. 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. D, WI-38 fibroblasts were transfected with either control (Ctl) or Cdc20 siRNA in the presence of either 1 mM or 10 mM lithium chloride (LiCl). Treatment with water (H₂O) served as control. After 1 day, cells were collected and the expression of Cdc20 and securin was determined by immunoblotting analysis using anti-Cdc20 and anti-securin IgGs. Ponceau S staining shows equal total protein loading. Quantification of P-securin is shown at the bottom of the blot. E, WI-38 cells were transfected with Cdc20 siRNA. Transfection with control (Ctl) siRNA was used as control. Cells were cultured for 3 days in the presence of either MG-132 (10 μM) or LiCl (10 mM). Treatment with DMSO served as control. Cells were collected and cell lysates were subjected to immunoblotting analysis with an antibody probe specific for securin. Ponceau S staining shows equal total protein loading. Quantification of protein band intensity is shown at the bottom of the blot. F, WI-38 cells were transfected with Cdc20 siRNA. Transfection with control (Ctl) siRNA was used as control. Cells were cultured for 10 days in the presence of LiCl (10 mM). Treatment with DMSO served as control. Cells were collected and cell lysates were subjected to immunoblotting analysis with an antibody probe specific for securin. Ponceau S staining shows equal total protein loading. Quantification of protein band intensity is shown at the bottom of the blot. DMSO, dimethyl sulfoxide.