JNK phosphorylation induced by heme is involved in macrophage cell death. (A) Macrophages from C57Bl/6 mice were stimulated with heme (30μM) in the time intervals indicated. (B) Macrophages were left untreated or pretreated for 1 hour with NAC at 10mM or 2 hours with DFX at 2mM and stimulated with heme (30μM). Cell extracts were submitted to SDS-PAGE and JNK phosphorylation was detected by immunoblotting. Detection of β-actin was used as loading control. (C) BMM (2 × 10⁵/well) from Tlr4^−/− mice were left untreated (−) or pretreated for 1 hour with SP600125 20μM and stimulated for 6 hours with heme at 30μM, and/or TNF at 0.5 ng/mL (+) and supernatants were collected for LDH determination. Data are representative of 3 independent experiments performed in triplicates and represent mean ± SEM (***P ≤ .0001). (D) Macrophages (10⁶/mL) were pretreated as in (C) and stimulated for 1 hour with heme (30μM) and ROS generation was evaluated. The results are representative of 3 different experiments with similar results.