Effects of inducibly expressed dn-p85^PI3-K on factor-independent growth of Ba/F^Val814 cells.

(A) Ba/F^WT cells and Ba/F^Val814 cells that inducibly express dn-p85^PI3-K by IPTG were preincubated with or without 1 mM IPTG for 24 hours at 37°C and then were stimulated with rmSCF (100 ng/mL) or medium alone for 15 minutes. Expression of mRNA was examined by Northern Blot analysis. (B) Ba/F3 cells that inducibly express dn-p85^PI3-K by IPTG were treated with 1 mM IPTG for the times indicated, and expression of dn-p85^PI3-K was examined by immunoblotting. (C) Ba/F^WT cells and Ba/F^Val814 cells that inducibly express dn-p85^PI3-K by IPTG were preincubated with or without 1 mM IPTG for 24 hours at 37°C and were then stimulated with rmSCF (100 ng/mL) or medium alone for 15 minutes. Cells were lysed, immunoprecipitated with antiphosphotyrosine Ab, and assayed for PI-3K activity. Individual spots represent phosphatidylinositol 3′-phosphate (PI-3P). (D) Ba/F^WT cells and Ba/F^Val814 cells that inducibly express dn-p85^PI3-K by IPTG were preincubated with or without 1 mM IPTG (control) for 24 hours at 37°C and were stimulated with rmIL-3 (10 ng/mL), rmSCF (100 ng/mL), or without either rmIL-3 or rmSCF, for 48 hours and then were subjected to [³H]thymidine incorporation. Three independent assays were performed with comparable results, and data from a representative experiment are shown. Bars represent standard errors. Statistically significant differences from control values are indicated by an asterisk (P < .01).