CO inhibits ER-stress–induced hepcidin expression via suppression of CREBH activation. (A) HepG2 cells were treated with TM (10 mg/mL) for the indicated times. (B) To detect cleavage and translocation of CREBH, cell lysates were divided into cytoplasmic and nuclear fractions after 12 hours of TM treatment, and each fraction was probed with CREBH-N Ab. Lamin A/C served as a marker for nuclear fractions. (C) Cells were incubated in the absence or presence of CORM-2 (20μM) and treated with TM for 12 hours to detect CREBH cleavage. (D) CREBH mRNA levels were analyzed by RT-PCR. (E-G) HepG2 cells were transiently transfected with scramble (SC) siRNA or CREBH siRNA and treated with TM and thapsigargin (TG) for 5 hours (E) or 18 hours (F), and hepcidin expression was analyzed for mRNA (using RT-PCR) and protein levels. (G) Transfected cells were treated with CORM-2 or DMSO, and incubated for 18 hours to induce hepcidin expression. 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.