Effects of mTORC2 knockdown on HT29 and LS174T cell proliferation. (A) Knockdown of mTOR or rictor reduces the proliferation of HT29 and LS174T cells. HT29 and LS174T cells were infected with lentiviral particles containing scramble, raptor, rictor or mTOR shRNA. Forty-eight hours post-infection cells were selected for resistance to puromycin for an additional 48 hours. Subsequently, 5 × 103 cells were plated on 96 well plates and cultured in DMEM 10% FBS for 2 or 5 days. Cell proliferation was determined by 3H-thymidine incorporation in the final 12 hours of cell culture. Results are expressed as the mean cpm ± standard deviation of three experiments. *p < 0.05 and **p < 0.01 compared with cells expressing the scramble shRNA (Student's t test). (B) Knockdown of mTOR or rictor induces a G1 arrest in HT29 and LS174T cells. HT29 and LS174T cells were processed as in A. Following selection for resistance to puromycin cells were cultures for 2 days and subsequently collected by trypsin digestion, washed in PBS incubated for 24 hours in 70% ethanol. Cells were subsequently resuspended in PBS containing 20 μg/ml propidium iodide and 200 μg/ml RNAse and incubated for 45 minutes at 37°C. DNA content of the cells was analysed with a FACScan II and the CellQUEST software (Beckton Dickinson). One of three similar experiments is shown.