Figure 3. Cyclin E mediates PKCα induction of DNA synthesis in SQ20B cells.

A) Effect of siRNA on DNA synthesis. SQ20B cells were transfected with control, PKCα, cyclin E, or E2F2 siRNA and protein expression was determined as described in Experimental Procedures (left panel). SQ20B cells were incubated in serum-free media (starved), serum alone (serum), serum with control siRNA, serum with 100nM Gö6976 (Gö6976), or serum with respective siRNAs for 24 hours (right panel). DNA synthesis was quantitiated for BrdU incorporation as described in Experimental Procedures and expressed as fold change compared with serum alone. B) Rescue of DNA synthesis with cyclin E and E2F2 expression in Gö6976 treated cells. SQ20B cells were mock transfected or transfected with cyclin E and E2F2 expression vectors and protein expression was determined as described in Experimental Procedures (left panel). SQ20B cells were incubated in serum-free media (starved), serum alone (serum), serum with Gö6976 (Gö6976), or serum with 100nM Gö6976 and the respective transfection vector(s) for 24 hours. DNA synthesis was quantitiated for BrdU incorporation as described in Experimental Procedures and expressed as fold change compared with serum alone (right panel). All BrdU incorporation results are the mean +/- S.E. of three independent experiments. C) Change in rate of cyclin E protein synthesis. SQ20B cells were either untreated or pretreated with Gö6976 (100 nM) for 12h and then exposed to MG132 (30 μM) for the times indicated. Graph represents quantification of cyclin E levels from the protein immunoblot. Cyclin E values were normalized to α-tubulin levels. Results are representative of three independent experiments.