Synergistic induction of target genes by ATRA and TNF. (A) Northern blot of BCL-3 and Dif-2. Total RNA (10 μg) was loaded in each lane. Ribosomal RNA levels indicate equal loading. BCL-3 mRNA levels were seen to increase after 2 hours of RA treatment in NB4 cells, whereas neither RA nor TNF alone had a significant effect on Dif-2 mRNA levels. mRNA levels of both genes were strongly induced on combined treatment with TNF and RA. (B) RT-PCR analysis of A20 RNA levels in response to ATRA. RT-PCR confirmed array results in that A20 levels decreased after 2 hours of ATRA treatment. —indicates no treatment. (C) Cytofluorometric analysis of TNFR2 levels on NB4 cells in response to 72-hour treatment with ATRA and TNF. Synergism between ATRA and TNF could be observed at 48 and 72 (data not shown) hours after treatment, whereas little effect could be seen at 2 hours. Percentages represent the number of positive cells that show FITC-positive staining.