(A) Rate of transcription of C/EBPε in untreated NB4 cells (control) and NB4 cells induced for 12 hours with 9-cis RA (5 × 10−7 mol/L). Nuclei were isolated for run-off experiments as described in the Materials and Methods. Equivalent counts of 32P-labeled RNA transcripts were hybridized to plasmids immobilized on nitrocellulose membranes as indicated (MPO, full-length MPO cDNA in PUC vector; pcDNA, empty pcDNA vector; C/EBPε, full-length C/EBPε antisense cDNA in pcDNA vector; β-actin, 3′ UT β-actin in PBR322 vector). (B) Half-life of mature C/EBPε RNA in NB4 cells. Northern blot of total RNA (30 μg/lane) of NB4 cells exposed for 0 to 4 hours to actinomycin (10 μg/mL) with or without prior treatment with 9-cis RA as indicated. (C) Effect of a protein synthesis inhibitor on C/EBPε RNA expression. Northern blot with total RNA (30 μg/lane) of NB4 cells treated either with 10 μg/mL CHX alone, with CHX (10 μg/mL) and 9-cis RA (5 × 10−7 mol/L), or with 9-cis RA (5 × 10−7 mol/L) alone. Cells were harvested at 0, 4, 6, and 8 hours. The top panel shows the hybridization with full-length C/EBPε cDNA probe. The bottom panel shows the hybridization with β-actin probe as assessment of RNA quantities on each lane. Fold-inductions were calculated by the ratio of densitometry readings of C/EBPε to β-actin. (D) Effect of ATRA on C/EBPε protein expression in NB4 cells. NB4 cells were cultured with ATRA (5 × 10−7 mol/L) for different durations. Lysates were made from the cells and Western blotted. COS cells transfected with either a human C/EBPε cDNA expression vector (lane +) or empty vector (lane −) were used as positive and negative controls, respectively. The arrow denotes C/EBPε protein as seen by Western blot.
