Figure 1

Characterization of ID8-NGL cells. (A) Schematic diagram of the pEGFPluc NGL reporter plasmid. The plasmid contains 4 tandem copies of a 36 base pair enhancer of the HIV long terminal repeat, which contains 2 consensus NF-κB binding sites (blue). Basal NF-κB reporter activity in ID8-NGL cells compared to parental ID8 cells was measured in (B) luciferase assays from protein extracts and (C) by bioluminescence imaging of cell monolayers. (D) Western blot showing GFP expression in ID8-NGL cells not observed in parental ID8 cells. Actin was used as the loading control. (E) Stimulatory effect of a 4 h treatment with TNF-α and IL-1β (both 10 ng/ml) on luciferase activity in protein extracts from ID8-NGL cells. (F) Western blot showing the stimulatory effect of TNF-α on GFP and p-IκB expression after 4 h is inhibited by 2 hours’ pre-treatment with the NF-κB inhibitor, thymoquinone (50 μM). Bioluminescent imaging is also shown. (G) Stimulatory effect of TNF-α on luciferase activity was also inhibited by thymoquinone (50 μM). Values are mean + SD of 3 independent experiments. * p < 0.01 relative to Control, # p < 0.01 relative to TNF alone, both Student’s t test.