Kinetics of NF-κB activation.

<p>TNFAIP3-luc2 reporter transfected mice were challenged with LPS (1 mg/kg, intratracheally, n = 11) or TNFα (1 µg/mouse, intravenously, n = 4) or saline (n = 4). Mice were imaged at 0 (prior to injection), 3, 6, and 24 hours after injection. Bioluminescent images from one representative mouse per group (A) and quantification of lung signals (B) are shown. Data presented as mean ± SEM.</p

Effect of anti-inflammatory agents on NF-κB induction in vitro.

<p>The effect of MLN120B (A) and TDZD-8 (B) on luciferase induction by LPS in TNFAIP3-luc2, NF-κB2-luc2 and IL8-luc2 transfected RAW264.7 cells are shown. All the compounds were tested at 1–100 µM concentrations. The data are presented as percentage change over vehicle treated cells. Data presented as mean ± SEM; n = 4 wells per reporter construct and LPS concentration.</p

In vitro comparison of NF-κB reporters.

<p>RAW264.7 cells were transiently transfected with NF-κB reporters. Cells were treated with LPS at 0–1 µg/ml concentrations overnight and imaged with IVIS after adding luciferin. Quantification of luciferase signal (A). Fold change of cells treated with 1 µg/ml LPS compared to vehicle treated cells (B). Data presented as mean ± SEM; n = 4 wells per reporter construct and LPS concentration of 1 µg/ml.</p

Effect of glycogen synthase kinase beta inhibition on NF-κB induction in vivo.

<p>NF-κB2-luc2 reporter transfected mice (n = 6 per group) were pre-dosed with either TDZD-8 (10 mg/kg, i.p.), or DMSO vehicle control 16 and 1-hour prior to LPS challenge. Mice were imaged immediately before LPS injection (T = 0) and at 3 hours post injection. Another group of mice was not treated with LPS and served as negative controls (n = 3). Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p

Effect of IKK2 inhibition on NF-κB induction in vivo.

<p>At 2 weeks after in vivo gene delivery with the NF-κB2-luc2 reporter, mice (n = 7) were pre-treated with MLN120B at 300 mg/kg orally or vehicle control 16 and 1-hour prior to LPS delivery. Mice were imaged immediately before LPS injection (T = 0) and at 4 hours. Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p