Figure 4.

(A) Chemokine mRNA levels. Ribonuclease protection assay (RPA) was performed using probes specific for the mRNAs encoding chemokines RANTES, IP10, MIP1β, MIP1α, MCP1, IL8, and for the control L32 and GAPDH transcripts. Total RNA samples were used from cells treated with PMA for the indicated times. (B) Cotransfection of C/EBPβ and TIF1β expression plasmids with the 8XC/EBPβ-luciferase reporter plasmid into U937 cells followed by a 24 h PMA differentiation. (C) Cotransfection of C/EBPβ and TIF1β expression plasmids with the −205 HIV–LTR-luciferase reporter plasmid in U937 cells followed by a 24 h PMA differentiation. (D) Cotransfection of C/EBPβ and TIF1β expression plasmids with −205 HIV–LTR-luciferase reporter plasmids with mutations in the two C/EBPβ binding sites (−205 HIV LTR m2m3) or in the two NF-κB binding sites (−205 HIV LTR mκB) in U937 cells followed by a 24 h PMA differentiation. The absolute luciferase values were 250,000 light units with C/EBPβ alone, and 1.5 × 10⁶ light units with both TIF1β and C/EBPβ cotransfected. (E) Transfections of U937 stable cell lines expressing β-gal and TIF1β antisense-ribozymes with increasing amounts of the 8XC/EBPβ-luciferase reporter plasmid followed by a 24 h PMA differentiation. (F) Transfections of U937 stable cell lines expressing β-gal and TIF1β antisense-ribozymes with increasing amounts of the −205 HIV–LTR-luciferase reporter plasmid followed by a 24 h PMA differentiation.