MEK inhibitor PD98059 blocks SCF-induced HbF production. (A) HPLC was performed on 1.5 million cells grown for 14 days in EPO-containing medium (4 U/mL) or EPO+SCF–containing medium (50 ng/mL), treated with increasing concentrations of various inhibitors. The HbF/HbA+HbF ratios are expressed as a percentage for EPO+SCF (upper solid lines) and EPO (lower dashed lines) for each inhibitor and shown with standard deviation bars (experiments performed in triplicate with cells from 3 separate donors). Points on the x-axis represent final concentrations for each inhibitor defined by (1:5) serial dilutions from a maximum concentration (Max) compared with cultures containing no inhibitors (0). Symbols and maximum concentration: AG490 (asterisk), max = 4 μM; LY83583 (plus sign), max = 125 μM; SU6656 (bar), max = 500 nM; NS2028 (square), max = 62.5 μM; GF109203X (cross), max = 1 μM; ODQ (diamond), max = 250 μM; SB203580 (circle), max = 2 μM; and wortmannin (triangle), max = 40 nM. (B) PD98059 effects on HbF/HbA+HbF. The format is the same as for panel A, except that the exact concentrations of PD98059 are shown for each serial dilution on the x-axis. P values demonstrating a significant reduction (P < .05) at 4 μM and 20 μM are shown and represent a comparison with cells cultured in the absence of PD98059. (C) PD98059 effects on cell count shown for comparison. (D) HPLC patterns showing hemoglobin peaks (mV) (HbF peaks marked by asterisk) of a representative experiment. E+S indicates culture containing EPO+SCF without PD98059; E+S+4 μM indicates culture containing EPO+SCF plus 4 μM PD98059; and E only, culture containing EPO alone (ie, without PD98059).