Hepcidin generated by hepatoma cells inhibits iron export from co-cultured THP1 monocytes  Bill Andriopoulos, Kostas Pantopoulos  Journal of Hepatology  Volume 44, Issue 6, Pages 1125-1131 (June 2006) DOI: 10.1016/j.jhep.2005.10.025 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Kinetics of 59Fe release from THP1 monocytic cells. The cells were loaded with 5μM 59Fe–Tf for 16h and the release of 59Fe in serum-free media was measured at the indicated time intervals. The media contained 100μM HMW-DFO to capture and solubilize 59Fe. Iron release is expressed as percentage of the total amount of soluble 59Fe in media divided by 59Fe in both media and cells. Values correspond to triplicate experiments (mean±SD). [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 The expression of hepcidin from Huh7 hepatoma cells inhibits 59Fe release in THP1 monocytes. (A) Huh7 hepatoma and H1299 lung cancer cells were treated with 20ng/ml IL-6 for 2h and the expression of hepcidin mRNA was analyzed by Northern blotting. (B) Huh7 cells were treated with 20ng/ml IL-6 and the expression of hepcidin (top) and β-actin (bottom) mRNAs at the indicated time intervals was analyzed by Northern blotting. (C) THP1 monocytes were loaded with 5μM 59Fe–Tf for 16h and co-cultured for 4h with H1299 or Huh7 cells, previously treated with 20ng/ml IL-6 for 2h or not. The release of 59Fe was monitored in serum-free media containing 100μM HMW-DFO. Iron release, calculated from the total amount of soluble 59Fe in media divided by 59Fe in both media and cells, is expressed as percentage of control (co-culture with H1299 cells). Values correspond to triplicate experiments (mean±SD). *p<0.05 versus control (one-way ANOVA test). Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 The expression of hepcidin from HepG2 hepatoma cells inhibits 59Fe release in THP1 monocytes. (A) Northern blot analysis of hepcidin (top) and β-actin (bottom) mRNAs in parent HepG2 cells and in HepG2 #6, stably transfected with a hepcidin cDNA. (B) THP1 monocytes were loaded with 5μM 59Fe–Tf for 16h and co-cultured for 4h with either control H1299 lung cancer cells, parent HepG2 cells, or HepG2 #6. The release of 59Fe was monitored in serum-free media containing 100μM HMW-DFO. Iron release, calculated from the total amount of soluble 59Fe in media divided by 59Fe in both media and cells, is expressed as percentage of control (co-culture with H1299 cells). Values correspond to triplicate experiments (mean±SD). *p<0.05 versus control (one-way ANOVA test). Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 The generation of hepcidin by Huh7 hepatoma cells decreases IRP2 levels in THP1 monocytes. THP1 monocytes were co-cultured for 4h with either control H1299 lung cancer cells, Huh7 cells, or Huh7 cells previously treated with 20ng/ml IL-6 for 2h. The expression of IRP2 (top) and β-actin (bottom) in THP1 cells was analyzed by Western blotting. IRP2/β-actin ratios were quantified by densitometry. *Denotes an apparently non-specific band. Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 The generation of hepcidin by Huh7 hepatoma cells stimulates ferritin synthesis in THP1 monocytes. THP1 monocytes were co-cultured for 2h with either control H1299 lung cancer cells, or Huh7 cells, or Huh7 cells previously treated with 20ng/ml IL-6 for 2h. Subsequently, the co-cultured cells were metabolically labeled with (50μCi/ml) trans-[35S]label for 2h. Lysates from THP1 cells were subjected to quantitative immunoprecipitation with ferritin antibodies. Immunoprecipitated material was analyzed by SDS-PAGE on a 15% gel and newly synthesized ferritin (arrow) was visualized by autoradiography. The relative band intensities were quantified by phosphorimaging. Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Image 1 Suppl. Fig. 1. Pre-treatment of control H1299 cells with IL-6 does not affect IRP2 expression (A) and ferritin synthesis (B) in co-cultured THP1 monocytes. Experimental conditions were as described in Figs. 4 and 5, respectively. Journal of Hepatology 2006 44, 1125-1131DOI: (10.1016/j.jhep.2005.10.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions