G-CSFR gene expression in NB4 cells treated with ATRA. (A) Total RNA (10 μg/lane) was isolated from NB4 cells induced to differentiate with ATRA (5 μM) for 0, 24, 48, and 72 hours and subjected to Northern analysis. Blots were sequentially probed with 32P-labeled cDNA probes for human G-CSFR, human C/EBPϵ, and rat C/EBPα. To monitor equal loading of RNA in each lane, the blot was probed with human γ-actin. (B) Northern analysis was performed on NB4 cells after short term (0.5, 1.5, and 3.0 hours) exposure to ATRA (5 μM), in the presence (a/c) and absence (a) of cycloheximide (10 μg/mL). As a control, NB4 cells were exposed to cycloheximide alone (c) in parallel. Total RNA (10 μg/lane) was isolated and subjected to Northern analysis as in 4A. (C) Chromatin immunoprecipitation (ChIP) analysis of C/EBPϵ and C/EBPα binding to the G-CSFR promoter. ChIP was performed from uninduced (0) and ATRA-induced (48 hours) NB4 cells using antibodies specific for C/EBPϵ (ϵ) and C/EBPα (α; ii) or preimmune serum (PIS) and a no-antibody control (–; i). The precipitated chromatin was analyzed using primers specific for the G-CSFR gene C/EBP site. Total input DNA (In; 1:10 dilution) was used as a positive control (i).
