FIG. 3.

TNF promoter activity in naive and LPS-tolerant Mono Mac 6 cells. Mono Mac 6 cells were transfected, cultured for 2 days with or without LPS (20 ng/ml), washed, and stimulated with LPS at 1 μg/ml. RNA and plasmid DNA were isolated; the plasmid DNA was amplified directly (30 cycles), while mRNA was reverse transcribed and amplified by using the 3′ transintron primer together with the 5′ primer (40 cycles). Products were separated on a 1.4% agarose gel (A). Densitometry values for mRNA are expressed as proportions of the plasmid DNA are 2.5, 65.9, 7.6, and 24.0% for lanes 1 to 4. Cell samples from the same experiment were taken for three freeze-thaw cycles, and luciferase enzyme activity was determined in the lysates (B). Values for luciferase were 5,218, 98,742, 9,623, and 21,931 relative light units for lanes 1 to 4. Data are representative of one experiment of three performed. The average fold induction for both mRNA and protein was significantly lower in LPS-stimulated tolerant cells than in LPS-stimulated naive cells. For mRNA, levels of induction were 39.8- and 4.4-fold in naive and tolerant cells, respectively; for luciferase protein, they were 13.9- and 2.1-fold for naive and tolerant cells, respectively (P < 0.05 for both mRNA and protein).