Fig. 1.

Effects of partial reduction of PRKDC on TNF-α-induced ICAM-1 expression in human cells and oxazolone-induced contact DTH or LPS-mediated ALI in mice. A Immunoblot analysis of protein extracts from HCT116 cells of different genotypes with antibodies against PRKDC or actin. B Cells were treated with TNF-α (10 ng/ml) for 24 h; protein extracts were assessed for ICAM-1. C Cells were treated as in (B) but for 6 h. RT‒PCR with primers specific to human icam-1 or gapdh. D Endothelial cells were treated with TNF-α with or without NU7441. Eighteen hours later, protein extracts were assessed for VCAM-1 or actin. E WT and PRKDC^+/− mice were sensitized to oxazolone on day 1. Challenge was performed on day 6 through application of 20 μl of 3% oxazolone in acetone to both ears. NU7441 (5 mg/kg) was administered after challenge. Ear thickness measurements were then measured. Edema was calculated for each ear as the difference in thickness before treatment (0 h) and 48 h after challenge. F Mice were administered, i.t., 10 μg/kg LPS or saline. Some WT mice received NU7441 i.p. after LPS exposure. Mice were sacrificed 18 h later. Cells of BAL fluids were differentially stained, and total cells, neutrophils (Neu), macrophages (MQ), and lymphocytes (Lym) were counted. G Lung homogenates were measured for myeloperoxidase (MPO). H Pulmonary edema was measured using the weight/dry ratio. Cytokine levels were measured in BALF (I) or sera of animals (J) using Multiplex kits. Data are expressed as the means ± SDs of values from n ≥ 6 per group. *, **, ***, difference from LPS-treated WT animals with p ≤ 0.05, p ≤ 0.01. p ≤ 0.01, respectively. K Total RNA from whole lungs was subjected to reverse transcription followed by real-time PCR with primers specific to mouse vcam-1. Data are means ± SDs of values from triplicates. *, **, ***, difference from TNF-α-treated cells with p ≤ 0.05, p ≤ 0.01. p ≤ 0.01, respectively