Fig. 3.

The BMP/SMAD pathway is involved in HCV core-mediated regulation of HAMP gene expression. a HCV core mediates changes in localization and expression of SMAD4 protein that is blocked by the BMP/SMAD inhibitor Dorsomorphin. Cytoplasmic (a1) and nuclear (a2) fractions of pTRE and C2-3 cells, as well as whole-cell extracts (a3), treated with 10 μM Dorsomorphin or vehicle control, were subjected to immunoblotting with an anti-SMAD4 antibody. β-actin and PARP1/2 were used as loading controls. b HCV core-mediated increase in b1 HAMP mRNA levels is abolished by the use of BMP/SMAD inhibitors. Following overnight starvation with 0.5 %(v/v) FCS, pTRE, and C2-3 cells were treated with 10 μM Dorsomorphin (D) or vehicle control and cells were harvested for RNA isolation and qRT-PCR with HAMP-specific primers. b2 HAMP protein levels return to normal following incubation with the BMP/SMAD inhibitor dorsomorphin. Supernatants from the experiment described in a1 were used for HAMP protein determination with a competitive ELISA. c SMAD4 is involved in the HCV core-mediated modulation of the HAMP gene promoter. Huh7 cells were co-transfected with the full-length and truncated HAMP promoter reporter constructs, expression plasmid for core or empty vector and a dominant negative construct for SMAD4 at various ratios. Promoter activity was measured using a commercially available luciferase assay