Figure 5: Assessment of apoptosis in Plk1-depleted primary mammalian cells.
(a) (Upper) Correlation of the Dox (0–1,000 ng ml−1)-induced knockdown of the Plk1 protein and the induction of apoptosis in lysates of wt and transgenic ES cells treated with Dox for 46 or 70 h are shown. The immunoblot for Plk1, caspase-3, PARP and β-actin is also presented. (Lower) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration). (Upper) Lysates of Plk1 siRNA-transfected (b) HUVEC, (c) fibroblasts and (d) keratinocytes were immunoblotted for Plk1, PARP and β-actin. Camptothecin (Camp) and/or nocodazole (Noc) controls are included. (Middle) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration). (Lower) Annexin V-based measurements of cells treated with siRNA Plk1 or a siRNA control (siRNA MM) were correlated with levels of apoptosis. (Upper) Lysates of siRNA (Plk1 and/or p53)-transfected (e) keratinocytes and (f) HUVEC cells were immunoblotted for Plk1, p53, p-p53(S15) and β-actin. Camptothecin (Camp) controls are included. (Lower) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration).
