Lentiviral introduction of Bcl-3 results in reduced LPS-induced TNF-α production in macrophages. (A) Schematic drawings of the lentivirus vector containing a murine Bcl-3 or SOCS3 gene cassette. (B) RAW cells were infected with lentivirus expressing Bcl-3/GFP or SOCS3/GFP. After infection for 24 hours, RAW cells were washed and additionally incubated for 2 days. The cells were then stimulated with LPS (10 ng/mL) for 6 hours, intracellularly stained for TNF-α, and analyzed by flow cytometry. (C) GFP-positive cells were collected with FACS, and stimulated with IFN-γ (30 ng/mL) and LPS (10 ng/mL) for 24 hours. The concentration of TNF-α in the culture supernatants was determined by ELISA. Error bars indicate SD. (D) GFP-positive cells (GFP alone and Bcl-3/GFP) were purified and stimulated with LPS (100 ng/mL) for 4 hours, and then total RNA extracts were analyzed for the mRNA expression of TNF-α. Hybridization with the β-actin probe confirmed even loading of RNA in each lane. (E) GFP-positive cells were stimulated with IFN-γ (30 ng/mL) and LPS (10 ng/mL) for 24 hours. The IL-6 concentration in the culture supernatants was measured by ELISA. Error bars indicate SD. (F) RAW cells were infected with a lentivirus expressing IκBϵ/GFP. After infection for 24 hours, RAW cells were washed and additionally incubated for 2 days. The cells were then stimulated with LPS (10 ng/mL) for 6 hours and analyzed for intracellular production of TNF-α by flow cytometry.