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suppmat_1295_fig1.tif91KExperimental design for cell culture and the corresponding treatments. Hepatocytes were isolated by collagenase perfusion and cultured with William's medium E containing 5% FBS for two hours. Afterwards, the medium was replaced by fresh medium without FBS and the culture was maintained for 18 hours. After the removal of culture medium, L-NAME or PGE1 were added at 4 or 2 hours, before D-GalN, respectively. Hepatocytes were harvested at the selected time points after PGE1 or D-GalN treatment.
suppmat_1295_fig2.tif115KEffect of PGE1 on cytokine-induced NOS-2 mRNA expression in the presence or absence of D-GalN in cultured hepatocytes. PGE1 (1 mol/L) was administered 2 hours before cytokines (200 U/mL IL-1 and 200 U/mL IFN-g) or D-GalN (5 mmol/L) in primary cultures of rat hepatocytes. The measurement of NOS-2 mRNA expression was carried out 12 hours after treatments by RT-PCR. Two bands of 216 bp and 138 bp corresponding to b-actin and NOS-2 mRNA, respectively, were observed in agarose gels. The blots are representative of two independent experiments.
suppmat_1295_fig3.tif151KProposed mechanism of negative inhibition of NOS-2 gene expression by PGE1-induced NO production. Pre-administration of PGE1 to hepatocytes activates NF-kB translocation resulting in NOS-2 expression, mRNA synthesis, NOS-2 protein translation and NO synthesis from L-arginine. NO synthesized exerts a negative feedback regulation on NF-kB activation and further NOS-2 expression induced by D-GalN and/or cytokines.

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