Titration of the effects of LY294002 on the proliferation and survival of erythroid progenitor cells. Day 8 cells that contained 64% ± 6% erythroid progenitor cells were suspended at a concentration of 5 × 105/mL in 1.0 mL serum-free medium supplemented with 2 U/mL EPO with various concentrations of LY294002 as indicated. After incubation for 4 or 24 hours, the cells were collected and washed twice in IMDM containing 0.3% BSA and then plated into serum-containing fibrin clots with 2 U/mL EPO. After 7 days of incubation, the clots were fixed and stained with benzidine-hematoxylin. (A) Viability; (B) absolute number of viable cells; (C) percent expression for the number of erythroid colonies relative to the value in 0.1% DMSO as 100%. The mean ± SD of triplicates is shown for C. Cells incubated for 4 or 24 hours with 0.1% DMSO contained 69% ± 4% and 65% ± 9% erythroid colony-forming cells, respectively. *P < .05, ***P< .001, decrease v control.
