Fig. 12. hVps34 KD cells exhibit a marked reduction in growth rate. (A) Following 2 days of selection, control (black) and hVps34 KD (gray) cells were seeded in 35 mm dishes at an equal density of 50,000 cells/dish. At the indicated time points, triplicate dishes from each cell line were harvested and counted with a Coulter Z1 particle counter (mean±s.e.m.). (B) Control (black) and KD (gray) cells were seeded in 25 cm² flasks at 150,000 cells/flask. On the indicated days, triplicate flasks of each cell line were incubated with [³H]thymidine (1.0 µCi/ml) for 5 hours. Radioactivity incorporated into TCA-precipitable material was counted and normalized to total cellular protein (mean±s.e.m.). (C) Control (black) and KD (gray) cells were seeded in 60 mm dishes at 200,000 cells/dish. On the indicated days, duplicate dishes from each cell line were harvested and stained with annexin-V. Annexin-positive cells were counted using a Guava personal cytometer. Cells treated overnight with TNF- (white) served as a positive control for apoptosis.