FIGURE 3.

Knockdown of C/EBP-β expression does not affect NGAL expression. Untransfected A549 cells (−) or cells transfected with 40 nmol of two different siRNAs against C/EBP-β (siB1 and siB2) or control siRNA (cntl) and stimulated with IL-17 (200 ng/ml) and TNF-α (20 ng/ml) for 2 h were analyzed for mRNA (A) and protein expression (B). A, the amount of C/EBP-β mRNA, as determined by real time PCR, showed a 2–2½-fold induction for untransfected and Cntl siRNA-transfected cells relative to unstimulated cells. In contrast, cells treated with siB1 and siB2 showed a C/EBP-β mRNA level about 20% of that found in cells with Cntl siRNA after stimulation with the two cytokines. B, analysis of total cell lysates showed a similar strong reduction in the amount of C/EBP-β protein in siB1- and siB2-transfected cells. Both the 35- and 20-kDa forms of C/EBP-β was affected. C, a new series of experiments again showed a significant reduction of C/EBP-β mRNA as well as of the IL-6 mRNA in cells treated with siB1 and stimulated for 2 h with IL-17 and TNF-α, whereas the amounts of C/EBP-δ and IL-8 mRNAs were unaffected. D, at 24 h post-stimulation the levels of C/EBP-β and IL-6 mRNAs was still reduced in siB1-transfected cells, whereas the transcripts for C/EBP-δ, IL-8, and NGAL were unaffected by the treatment. E, this observation also holds true for NGAL, IL-6, and IL-8 also when measuring the amount of these three proteins in the medium of the cytokine-stimulated cells, as comparable levels were found for NGAL and IL-8 irrespective of whether the cells were transfected with siRNA or not, whereas the amount of secreted IL-6 was strongly diminished from siB1-treated cells. The real time PCR data are shown relative to the most highly expressed transcript in each experiment. Data are presented as the mean ± S.D. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.