Regulation of transcription factors involved in monocytic differentiation in RA-resistant APL cells. (A) Northern blotting of IRF-7 mRNA levels in RA-resistant APL cells. IRF-7 levels were seen to increase dramatically in cells treated with RA and TNF but not in response to either agent alone. The top panel for each cell line shows IRF-7 levels, and the bottom panel represents actin mRNA levels. (B) Western blotting showing c-jun expression in RA-resistant APL cells. Results show a positive correlation between c-jun expression and cells undergoing monocytic maturation. (C) Electrophoretic mobility shift assay (EMSA) analyses of AP-1 binding in RA-resistant APL cells in response to TNF and RA. Binding to an AP-1 site was seen to increase in response to these 2 agents in all cells tested except for UF-1. (D) EMSA analysis of PU.1 DNA binding in RA-resistant APL cells in response to RA and TNF at 72 hours after treatment. Binding data demonstrate an increased binding affinity of PU.1 in response to TNF and RA in cells undergoing monocytic differentiation. The presence of PU.1 in binding complexes was confirmed in reactions using anti-PU.1 antibodies. Complexes specifically inhibited by the PU.1 antibody are denoted with an arrow. Binding reactions were performed with a consensus PU.1 oligonucleotide and 12 μg nuclear extracts.