Two identified small molecules reduce NF-κB DNA binding activity in vivo and in vitro.

<p>(A) Levels of p-IκBα with or without treatment in response to TNF-α were determined by western blot analysis. HEK293 cells were pretreated with DMSO, ZINC12909780 (100 μM), and ZINC5 (100 μM) for 30 min followed by the stimulation of 10 ng/ml of TNF-α for 0, 5, and 10 min. Cell lysates were prepared for immunoblot against p-IκBα. GAPDH was used as a loading control. (B) EMSA analysis detecting NF-κB DNA binding activity was performed in C2C12 myoblasts. Cells were pretreated with 200 μM of DMSO, ZINC12909780, and ZINC5 in serum-free media for 1 h. TNF-α was then added to a final concentration of 10 ng/ml and incubated for 15 min. Nuclear extract was obtained for EMSA analysis. (C) EMSA analysis assessing NF-κB DNA binding activity in vivo was performed with quadriceps from mdx mice. Single-dose ZINC12909780 and ZINC5 at 10 mg/kg was given by i.p. Quadriceps was harvested at 1 h and 2 h post injection for EMSA analysis. (D) The time-course pharmacokinetics of ZINC5 and ZINC12909780 in FBS media or FBS media containing acetonitrile. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004663#pbio.2004663.s001" target="_blank">S1 Data</a>. EMSA, electrophoretic mobility shift assay; HEK293, human embryonic kidney 293 cells; i.p., intraperitoneal; NF-κB, nuclear factor κB; t_1/2, half-life; TNF-α; tumor necrosis factor α.