An active PI3K is required for the proliferation of HS1 and HS2 cells.
663 HS1 cells (A) or 606 HS2 cells (B) were cultured for the indicated times in the presence or absence of 10 μM LY294002. The means and standard deviations were determined from 4 experiments. (C) 663 HS1 cells were either grown in the presence of 2 U/mL (lane 663) or starved in Epo and serum for 4 hours before being stimulated for 30 minutes with 10 U/mL of Epo (lane 663stim). 606 HS2 cells were either grown with serum (lane 606) or stimulated for 30 minutes with 10 U/mL Epo and serum after 4 hours of serum starvation (lane 606stim). Anti-p85 immunoprecipitates from cell lysates were tested for PI3K activity in an in vitro PI3K assay using the phosphatidylinositol 4,5-bisphosphate as substrate. A control (lane C) is the PI3K activity in cell extracts immunoprecipitated with a nonimmune serum. The PIP3 was separated by thin-layer chromatography and visualized by a Phosphorimager.
