FIGURE 8.

PLK inhibition affects pro-inflammatory responses. A, quantitative RT-PCR analysis of RNA isolated from several tissues from C57/Bl6N mice. plk2 and adam17 expression levels were normalized against gapdh mRNA. The corresponding spleen signal was fixed at 100%, and all other signals were expressed as a proportion of this (n = 3). One representative result from three replicate experiments is shown. B and C, C57/Bl6N mice were challenged for 1 h with LPS. Expression levels of plk2 and adam17 mRNA was determined by quantitative RT-PCR (n = 2 untreated mice; n = 3 LPS-treated mice). D, BMDMs as well as BMDCs were seeded at a density of 1 × 10⁶/6-well and incubated for indicated time periods with 1 μg/ml LPS. plk2 mRNA was quantified by quantitative RT-PCR. One representative experiment is shown (n = 3). E and F, BMDMs and BMDCs were seeded as described in D. After 24 h, cell culture medium was replaced, and the cells were stimulated with 1 μg/ml LPS (12 h) in presence or absence of the metalloprotease or PLK inhibitors BI 2536 (BI, 1 μm), GI254023X (GI, 3 μm), and GW208264 (GW, 3 μm). Inhibitors were added 10 min prior to stimulation. Soluble TNFα (D and E) or sTNFRI (D) concentrations were determined by ELISAs in the supernatants (n = 3). All error bars denote S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. For the ELISA experiments, we show the means of three independent experiments performed in duplicate.