Impact of siRNA-mediated specific inhibition of BMP type II receptor expression on BMP signaling and hepcidin expression induced by HJV in Hep3B and KGN cells. (A) Expression of BMP type II receptors BMPRII, ActRIIA, and ActRIIB in Hep3B cells and human liver (RT-PCR). KGN cells were used as a positive control. (B-F) Hep3B (B-D) or KGN cells (E,F) were transfected with BRE-Luc (B,C,E,F) or the activin-responsive firefly luciferase reporter (CAGA-Luc; panel D) and pRL-TK Renilla luciferase vector in combination with control siRNA or siRNA specific for BMPRII, ActRIIA, or ActRIIB (60 nM). Cells were incubated in the absence or presence of 20 ng/mL BMP-2 (B,E) or activin A (D) or cotransfected with HJV cDNA (8 ng/mL; panels C,F). Cell lysates were analyzed for luciferase activity as in Figure 1B-E. (G,H) Hep3B cells were transfected with the hepcidin promoter reporter construct (Hep-Luc) and pRL-TK, in combination with control siRNA or siRNA specific for BMPRII, ActRIIA, or ActRIIB (40 nM). Cells were incubated in the absence or presence of 20 ng/mL BMP-2 (G), or after cotransfection with HJV cDNA (8 ng/mL; panel H) prior to measurement of luciferase activity. (I) Hep3B cells were transfected with empty vector (pcDNA3) and control siRNA, or with Flag-HJV cDNA in combination with control siRNA, si-BMPRII, si-ActRIIA, or si-ActRIIB at 40 nM. At 46 hours after transfection, hepcidin relative to RPL19 mRNA levels were quantified by real-time RT-PCR as in Figure 1G,H. Values shown are the means of triplicate measurements plus or minus SD. *P < .05; **P < .01; ***P < .001.