Figure 4.
Inhibiting the dTOR/dRaptor complex with rapamycin promotes progression from S phase through G2/M phase. (A) S2 cells seeded at 1 × 106 cells/ml were pulse-labelled with BrdU for 15 min and then grown in the presence or absence of 100 nM rapamycin and 1 μM insulin. The cell-cycle profiles of the BrdU-labelled cells fixed at the time points shown were recorded by flow cytometry. The ability of rapamycin to accelerate progression from S phase through G2 and M phases and into G1 is evident after 9 h and more marked by 12 h. Data are indicative of three independent experiments. (B) Cells were seeded at 1 × 106 cells/ml, then grown in the presence or absence of 100 nM rapamycin and 1 μM insulin for 9 h and immunostained with anti-phospho-H3. The percentage of cells in mitosis for each condition was determined by counting the number of phospho-H3 positive cells and total number of cells in six fields of view (n=at least 250). Insulin treatment reduces and rapamycin treatment increases the percentage of cells in M phase. Data shown represent mean data±s.d. from three replicates (*P<0.01) and are representative of two independent experiments.