Inhibition of ERK activity inhibits proliferation but does not induce apoptosis or JNK activation in HCD57 cells.

HCD57(R) cells were washed and treated with either EPO (EPO, lane 2), EPO + DMSO vehicle (V, lane 3), EPO + 50 μmol/L PD98 059 (PD, lane 4), or no additional growth factor (No EPO, lane 1) for 48 hours. (A) Rabbit-anti-JNK-1 immunoprecipitates were subjected to an in vitro kinase assay using γ-³²P-ATP and GST-Jun fusion protein as a substrate and transferred to nitrocellulose. Following exposure to show the phosphorylated GST-Jun (upper arrow), the proteins were immunoblotted with goat-anti-JNK to visualize JNK proteins (lower arrows). (B) Rabbit-anti-ERK immunoprecipitates were immunoblotted with mouse-anti-phospho-ERK antibody to visualize phosphorylated ERK proteins (upper arrow). The blot was then stripped and reprobed with anti-ERK antibody to ensure equal loading of proteins (arrow, lower panel). (C) Proliferative response of HCD57(R) cells to EPO and PD98059 24 hours (columns 1-4) and 48 hours (columns 5-8) following addition of EPO (columns 2-4 and 6-8) or no growth factor (columns 1 and 5) in the presence of PD20859 (columns 4 and 8). Data are indicated as the number of cells as a percentage of the starting number of cells. In this experiment, nonviable cells were 5% or less. (D) Cells incubated for 48 hours with no additional growth factor (upper left panel), EPO alone (upper right panel), EPO + DMSO (lower left panel), or EPO + PD98059 (lower right panel) were stained with propidium iodide as indicated in the “Materials and methods” section and analyzed by using flow cytometry. The number of apoptotic cells is indicated as a percentage of the total number of cells containing sub-G₀/G₁ DNA and is indicated as M1 on plots.