Targeted gene silencing of MyD88-independent signaling pathway molecules TRIF or IRF3 in human macrophages reduces TLR4-mediated RANTES release (but not TNF release). RNAi-based targeted gene silencing of TRIF (A-D) or IRF3 (E-H) in human macrophages was performed as detailed in “Targeting gene silencing (RNAi) in macrophages.” (A) Western blot analysis of human U937 and HIV⁺ U1 macrophages after pretreatment with RNAi targeted to TRIF (TRIF siRNA) or nonsilencing RNAi (N.S. siRNA) and probed with anti-TRIF antibody. Anti–β-actin antibody was used to monitor protein loading. The blot is representative of 3 independent experiments with similar results. (B-D) Human macrophage U937 and U1 cells, pretreated with either TRIF siRNA or N.S. siRNA, were then incubated with or without lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for RANTES (B-C) or TNFα (D) by ELISA. Data shown are mean ± SEM of 3 independent experiments done in triplicate with similar results. (E) Western blot analysis of human U937 and U1 macrophages after pretreatment with RNAi targeted to IRF3 (IRF3 siRNA) or N.S. siRNA and probed with anti-IRF3 antibody. Anti–β-actin antibody was used to monitor protein loading. The blot is representative of 3 independent experiments with similar results. (F-H) U937 and U1 cells, pretreated with either IRF3 siRNA or N.S. siRNA, were then incubated with or without lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for RANTES (F-G) or TNF (H) by ELISA. Data shown are mean ± SEM of 3 independent experiments done in triplicate with similar results.