Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death
Figure 4
Inhibition of Cdk5 expression does not protect cells from death. (a) COS-7 cells transfected with dsRNA (Cdk5-1, Cdk5-2, Cdk5-3) were collected and lysed by RIPA buffer. Western blot of equal amounts of cell lysates from COS-7 cells showed Cdk5 expression in all cells (A), and the amount of Cdk5 was measured by densitometry (B). dsRNA of Cdk5-1 and Cdk5-3 downregulated Cdk5 expression. (b) COS-7 cells transfected with Cdk5-1, 5-2, or 5-3 dsRNA were treated with 25 M CPT for 48 hours, and cell death was measured by trypan blue exclusion. Downregulation of Cdk5 by RNAi did not affect the amount of cell death. The amount of cell death was calculated by subtracting the amount of cell death in untreated control cells. The error bars represent the standard deviation from at least three individual experiments. (c) COS-7 cells treated with 25 M CPT for 48 hours were fixed with 4% paraformaldehyde and stained with Hoechst 33 258 dye. Apoptotic cells manifest condensed and fragmented nuclei as is seen in (D), (E), and (F). (A) Control cells; (D) CPT-exposed control cells; (B) cells transfected with Cdk5-1; (E) cells transfected with Cdk5-1 and treated with CPT; (C) cells transfected with Cdk5-2; (F) cells transfected with Cdk5-2 and treated with CPT. The numbers and percent of dead cells were not calculated but appeared to be consistent with the results presented in Figure 4(b). (d) COS-7 cells were incubated with 25 M CPT for 48 hours with or without a dsRNA. After 48 hours incubation, cells were collected and proteins were extracted. Western blots of cell lysates from cells showed the activation caspase-3 by CPT both in the control cells and transfected cells.
