CO inhibits IL-6–induced hepcidin expression via suppression of CREBH activation. (A) HepG2 cells were incubated with IL-6 for increasing time intervals. (B) To detect the cleavage and translocation of CREBH, cell lysates were divided into cytoplasmic and nuclear fractions, and each fraction was probed with CREBH-N Ab. Lamin A/C served as a marker for nuclear fractions. (C) Cells were preincubated for 3 hours with CORM-2 (20μM) and then treated with IL-6 for 12 hours. CREBH-N was analyzed by Western blotting. (D) CREBH mRNA levels were analyzed by RT-PCR. (E-F) HepG2 cells were transiently transfected with scramble siRNA or CREBH siRNA, preincubated for 3 hours with or without 20μM of CORM-2, and then treated with IL-6 for 18 hours to induce hepcidin expression (F). To analyze HAMP mRNA level, cells were incubated with IL-6 for 4 hours (E). β-actin served as the standard for Western blots. GAPDH served as the standard for mRNA. Values are means ± SEM from 3 independent experiments. *P < .05; **P < .01;