Figure 1.

A/ Growth rate and survival of RAW 264.7 cells cultured in medium, or medium supplemented with LPS or IFN-γ or LPS+IFN-γ. RAW 264.7 cells were cultured (4 × 10⁶ cells in 20 ml) in medium (• œ) or medium supplemented with either IFN-γ (50 U/ml -.), or LPS (5 μg/ml -) or a mixture of IFN-γ (50 U/ml) + LPS (5 μg/ml -). The number of live cells recovered at the indicated times was estimated using trypan blue exclusion. Similar results were obtained estimating the cell survival either by 3H-thymidine incorporation, or by the MTT reduction test. B/ NOS2 (iNOs) mRNA induction in RAW 264.7 cultured with LPS or IFN-γ or LPS+IFN-γ. RAW 264.7 cells seeded into six-well plates (2.5 × 10⁶ cells/well) were treated with the different stimuli as indicated above for the indicated times and analyzed by RT-PCR with specific primers for murine NOS2 (iNOs) (Table 1). HPRT was used as internal control for semi-quantitative estimation. C/ NOS2 (iNOs) protein in RAW 264.7 cultured with LPS or IFN-ã or LPS+IFN-γ. RAW 264.7 cells were treated with the different stimuli as indicated above for various times and cell pellets (1 × 10⁶ cells) were lysed with lysis buffer. Protein concentrations in samples were adjusted and electrophoresed on 15% SDS-polyacrylamide gel, then transferred to nitro-cellulose membrane and Western bloted using polyclonal rabbit anti-murine NOS2 (iNOs), as described in Materials and Methods.