HB-EGF activity is mediated by activation of the PI-3K/AKT pathway. (A) XG-7 myeloma cells were starved overnight in culture medium without serum or cytokines. Cells were then stimulated with either IL-6 (20 ng/mL), IGF-1 (1 μg/mL), or HB-EGF (5 μg/mL) for 15 minutes at 37°C. Lysates from unstimulated (Co) or stimulated myeloma cells were immunoblotted with anti–phospho-specific STAT3 antibody (top panel) and then reprobed with anti-STAT3 antibody (second panel), anti–phospho-specific AKT antibody (third panel) and reprobed with anti-AKT antibody (fourth panel), anti–phospho-specific MAPK antibody (fifth panel) and reprobed with anti-MAPK antibody (bottom panel). (B) XG-5 and XG-7 myeloma cell lines were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with 5 pg/mL IL-6 and 1 μg/mL HB-EGF without (□) or with (▪) the PI-3K inhibitor LY294002 (25 μM). Cells were cultured for 6 days and pulsed with tritiated thymidine at the end of the culture. Mean tritiated thymidine incorporations were determined in sixplicate culture wells and results are expressed as percentages ± SDs of the mean proliferation without ErbB inhibitor. *Indicates that the mean value is statistically significantly different from that obtained with 5 pg/mL IL-6 and HB-EGF without inhibitor, using a Student t test (P ≤ .05).