Figure 4.

Effect of nepalolide A on the level of iNOS. C6 glioma cells (a) and astrocytes (b) were preincubated with nepalolide A (2–10 μM) for 30 min. After 30 min incubation, C6 glioma cells were challenged with 5 μg ml⁻¹ LPS/100 units ml⁻¹ IFN-γ (LPS/IFN-γ) or 5 ng ml⁻¹ TNF-α/5 ng ml⁻¹ IL-1β/100 units ml⁻¹ IFN-γ (mixed cytokines) plus or minus nepalolide A for 24 h. For astrocytes, cells were challenged with 5 ng ml⁻¹ TNF-α/5 ng ml⁻¹ IL-1β/100 units ml⁻¹ IFN-γ in the presence or absence of nepalolide A for 24 h. After incubation for 24 h, cells were harvested and iNOS was immunoprecipitated and analysed by immunoblotting. The top parts of (a) and (b) are immunoblots of iNOS. The bottom parts are the relative levels of iNOS in untreated cells and cells treated with nepalolide A. Results are means±s.d. (where large enough to be shown) from four independent experiments, and are expressed relative to cells treated with iNOS inducers alone. Significant differences between control and nepalolide A-treated cells are indicates by ^*P<0.05; ^**P<0.01; and ^***P<0.001.