Fig 1: Detection of TNF-a and IL-6 levels using an enzyme-linked immunosorbent assay. a: Detection of IL-6 levels. b: Detection of TNF-a levels. Note *P < 0.05, **P < 0.01 vs the MCs group, #P < 0.05, ##P < 0.01 vs Ad-CREBHi or Ad-USi. MCs: myocardial cells, H/R: hypoxia/reperfusion, Ad-USi: adenovirus for the unspecific, Ad-CREBHi: adenovirus for cyclic AMP-responsive element-binding protein H (CREBH) RNAi, TNF-a: tumor necrosis factor-a, IL-6: interleukin-6.
Fig 2: Changes of signaling proteins responsible for hepcidin expression after GCDCA treatment in HepG2 cells. (A) Representative western blotting bands shown in the left side of panel of A display the phosphorylation of SMAD1/5/8 (normalized by total SMAD1) and STAT3 (normalized by total STAT3) in response to different GCDCA treating time. Fold changes in optical density relative to controls are shown on the right side of panel of (A). (B) The phosphorylated and total protein levels of both SMAD1/5/8 and STAT3 after 2 h treatment with GCDCA. (C) The phosphorylated and total levels of SMAD1/5/8, STAT3 and CREBH in the nucleus after a 2-h treatment with GCDCA. (D) The hepcidin gene expression under co-treatment of 200 µM GCDCA and 150 nM LDN-193189 (SMAD inhibitor) for 2 h. (E) Representative western blotting bands showed the phosphorylation levels of SMAD1/5/8 under co-treatment of GCDCA and LDN-193189. Values were shown as means ± SD. *, p < 0.05, GCDCA treatment group vs. control group. **, p < 0.01. ††, p < 0.01, other experimental groups vs. control group. ##, p < 0.01, GCDCA treatment group vs. other experimental groups. Each experiment was performed at least three times.
Fig 3: GRP78 and CREBH protein expression using western blotting. a: Western blotting results of GRP78 protein expression levels. b: Statistical analyses of results shown panel “a”. c: Results of western blotting of CREBH protein expression levels. d: Statistical analyses of results shown in panel “c”. e: Results of western blotting analyses of knockdown of CREBH protein levels in the hepatocytes using adenoviruses encoding CREBH-specific shRNA (Ad-CREBHi). f: Statistical analyses of results shown in panel “e”. Note: *P < 0.05 vs the MCs group, #P < 0.05 vs the L/M + Tm group, $P < 0.05, $$P < 0.01 vs CREBH-N.Tm: tunicamycin, H/R: hypoxia/reperfusion, MCS: myocardial cells, L/M: lower compartment and hepatocytes, Ad-USi: adenovirus for the unspecific, Ad-CREBHi: adenovirus for cyclic AMP-responsive element-binding protein H (CREBH) RNA.i
Fig 4: Effect of H/R treatment in co-cultured hepatocytes. a: Results of reverse transcription-polymerase chain reaction for GRP78, CHOP, CRP, and CREBH mRNA expression in hepatocytes stimulated by IL-6 (60 ng/mL) or TNF-a (20 ng/mL). b: Results of reverse transcription-polymerase chain reaction for GRP78, CHOP, CRP, and CREBH mRNA expression in hepatocytes after 12 hours of H/R stimulation in MCs. c: Western blotting results of GRP78, CHOP, CRP, and CREBH protein expression in hepatocytes. d: Fold activity of GRP78, CHOP, CRP, and CREBH protein expression in hepatocytes.H/R: hypoxia/reperfusion, CRP: C-reactive protein, CREBH: cyclic AMP-responsive element-binding protein H, TNF-a: tumor necrosis factor-a, CHOP: C/EBP homologous protein
Fig 5: CCK-8 test of the viability of MCs. a: Viability of MCs in the MCs + H/R, MCs + Tm, L/M + H/R, L/M + Tm, and untreated control groups. b: Viability of MCs with Ad-CREBHi and Ad-USi. Note: *P < 0.05 vs the control group.Tm: tunicamycin, H/R: hypoxia/reperfusion, MCS: myocardial cells, L/M: lower compartment and hepatocytes, Ad-USi: adenovirus for the unspecific, Ad-CREBHi: adenovirus for cyclic AMP-responsive element-binding protein H (CREBH) RNAi.
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