Activated RAW 264.7 macrophages provoke HIF-1α accumulation in LLC-PK₁ cells under coculture conditions.

RAW 264.7 macrophages were stimulated with vehicle (I), 1 μg/mL LPS in combination with 100 U/mL IFN-γ (II), LPS/IFN-γ plus 1 mM L-NAME (III), or LPS/IFN-γ plus 100 μM carboxy-PTIO (IV) for 18 hours. Afterward, macrophages were resuspended in fresh medium as described in “Materials and methods” and added to LLC-PK₁ cells in a ratio of 1:1 (A) or 1:2 (B) (LLC-PK₁:RAW 264.7). Cells were cocultured for 4 or 6 hours in the absence or presence of 1 mM L-NAME (III) or 100 μM carboxy-PTIO (IV). Cells were prepared for HIF-1α Western blot analysis, while medium was used for nitrite measurements. Results are representative for 3 different experiments.