Figure 5.

Ectopic expression of SIRT1 attenuates insulin-like growth factor-1 (IGF-1)-induced premature cellular senescence. IMR90 cells were infected with retrovirus expressing murine SIRT1 or vector control and selected by puromycin resistance. Stable cells were serum-starved for 4 days and then treated with 50 ng mL⁻¹ IGF-1 every other day for 6 days. (A) Cells were assayed for SA-β-Gal activity and photographed under a light microscope. (B) Senescent cells were quantified by counting the number of SA-β-Gal-positive cells over the total number of cells from four randomly selected fields of each cell culture plate. Results are presented as means and SE from two experiments performed in duplicate. (C) Whole-cell extracts were subjected to western blot analysis as shown. (D–F) IMR90 cells were stably transfected with lentivirus expressing human wild-type SIRT1 or point-mutant SIRT1(H363Y), or a vector control. Stable cells were serum-starved for 4 days and then treated with 50 ng mL⁻¹ IGF-1 every other day for 6 days. (D) Whole-cell lysates were subjected to western blot analysis. (E) Stable cells were assayed for SA-β-Gal activity and photographed under a light microscope. (F) Senescent cells were quantified as before. Results are presented as means and SE from two experiments. ***P < 0.001.