Fig. 4. Role of HBEGF shedding at 2% O₂. (A) Cellular and secreted HBEGF concentrations were measured by ELISA after culture for 8 hours at 2% O₂ in medium containing the indicated concentrations of GM6001. HBEGF values were normalized to vehicle, as in Table 1. ^*P<0.05 compared with vehicle. (B) Cytotrophoblast cells were cultured for 8 hours at ambient O₂ levels (black bars) or 2% O₂ (gray bars) in medium supplemented with vehicle, 10 µg/ml GM6001 or 10 µg/ml of an inactive structural analog (Control) and TUNEL was determined. ^*P<0.05 compared with vehicle treatment. (C) Cells were cultured for 8 hours at 2% O₂ with 0 µg/ml (x) or 10 µg/ml (all other symbols) GM6001 and apoptosis was determined by TUNEL. The indicated concentrations of recombinant human HBEGF (black squares) were added to GM6001-treated cells. Co-treatment was also conducted with 10 nM of other recombinant EGF family growth factors, including epiregulin (open circle), betacellulin (open diamond), amphiregulin (open triangle), EGF (open square) or TGF (solid triangle). The 0 µg/ml GM6001 control was different (P<0.05) from all other treatments except those containing 10 µg/ml GM6001 plus 1-10 nM HBEGF.