MyD88dn and Maldn block LPS-induced NF-κB activation and cytokine production in human SFs and HUVECs. SFs and HUVECs were infected with adenoviruses overexpressing β-galactosidase, MyD88dn, Maldn, or MalTIR in serum-free medium. Then cells were cultured in complete medium for another day. (A) Cells were examined for the expression of MyD88dn and Maldn. SFs (B) and HUVECs (C) were treated with 20 ng/mL IL-1, 1 μg/mL LPS, or 20 ng/mL TNFα for 20 hours, and supernatants from uninfected (white bars), Adβ-gal– (gray bars), AdMyD88dn- (black bars), AdMaldn- (cross-hatched bars), or AdMalTIR-infected (checkered bars) cells were assayed by ELISA for IL-6. Mean cytokine production (± SD) of triplicate cultures is shown and is representative of 5 independent experiments from unrelated donors. (D) SFs were stimulated for 45 minutes with 20 ng/mL IL-1, or 1 μg/mL LPS; cytosolic and nuclear extracts were collected and then examined for IκBα expression by Western blot (upper panel) and NF-κB DNA binding by EMSA (lower panel).