SIRT2 stable transfectants that overexpress the wild-type phosphorylated forms of SIRT2 return to G1 less effectively than cells expressing mutant SIRT2 or nonhyperphosphorylated cell lines. Stable transfectants and the parental Saos2 cell lines were tested for their ability to exit mitosis and enter the cell cycle after release from nocodazole block in M. Cell lines were treated with nocodazole for 20 h, and then the nocodazole was washed out. Cells were collected for flow cytometry at the indicated times after the initiation of nocodazole washout. In panels A to E, graphs indicate the percentage of cells with a 2N and 4N DNA contents at each time point for each cell line.
Following removal of nocodazole from the medium, the parental cell line Saos2, H232Y-1, and H232Y-21 readily resumed the cell cycle and entered into G1, as evidenced by the increase in 2N DNA content after 6 h .Clone 4 and clone 2 overexpressed the phosphorylated SIRT2 isoforms. After 24 h, 65 to 80% of the cells had a 2N DNA content. In contrast, even 24 h after removal of nocodazole from SIRT2-overexpressing clone 2 and clone 4 cells, 60 to 65% of the cells remained in G2/M.
Cell lysates were harvested at each time point and immunoblotted with SIRT2 peptide antibody (results shown below each graph). UT, untreated, unsynchronized cells. These results are representative of those obtained in multiple experiments. The more hyperphosphorylated the cell line is, the slower the exit from mitosis.
