Figure 3. PKC_α is necessary and sufficient to affect cell cycle progression. A: Flow cytometry analysis of RPE cells after 100 nM thymeleatoxin treatment shows a cell cycle progression profile similar to that obtained with PMA in eight experiments. B: western blot analysis shows that PKC_α was rapidly translocated to the membrane by thymeleatoxin and downregulated within 24 h, the protein remained undetectable after 48 h of treatment, however, PKC_δ was not translocated and was not downregulated at all time points. Eighty micrograms of protein was loaded in each well. Optical density of PKC_α determined by densitometric imaging is shown (Mean±SD, n=3). The β-actin band with 42 kDa is used for quantitation. C: Flow cytometry analysis of RPE cells shows that there was no significant change in the cell cycle progression following PMA or thymeleatoxin restimulation when compared with the control over the 30 h time course after 48 h of PMA treatment. D: PKC_α activity regulates the growth rate of RPE cells. Approximately 110,000 RPE cells were seeded and then incubated with thymeleatoxin or siRNA-PKC_α for 24h. The numbers of cells were counted using a Coulter Counter and displayed in the top panel (* p<0.0001). Western blot using an anti-PKC_α antibody showed that the total PKC_α level was dramatically decreased in siRNA-PKC_α treated cells; 40 µg of protein was loaded in each well.