G-CSF regulates C/EBPε.
(A) Induction of C/EBPε by G-CSF. Total RNA extracted from 32Dcl3 cells (20 μg) treated with 50 ng/mL recombinant human G-CSF for the indicated times was subjected to Northern blot analysis. (B) Growth curve for 32Dcl3 cells cultured in various cytokines. The 32Dcl3 cells maintained in the presence of IL-3 were washed extensively with PBS and transferred to medium containing 25 U/mL IL-3 (●), 50 ng/mL G-CSF (▪), IL-3 and G-CSF (▴), or medium without cytokines (○). Viable cells were counted using the trypan blue dye exclusion method at the times indicated. Cells were diluted with each medium to keep cell density within 2 to 10 × 105/mL. (C) Expression of C/EBPε and C/EBPα in 32Dcl3 cells treated with IL-3, G-CSF, or both. Cells growing in IL-3 were washed twice with PBS and transferred to medium containing the indicated cytokines. Northern blot analysis was done as described above. (D) Comparison of expression of C/EBPε and C/EBPα in 32Dcl3 cells and NFS60 cells. Cells were starved in RPMI 1640 with 10% FBS lacking cytokines for 14 hours and then stimulated with 50 ng/mL G-CSF for the times indicated. Each total RNA (20 μg) was then analyzed by Northern blot analysis.
