Figure 1
Impact of siRNA-mediated specific inhibition of BMP ligand expression on BMP signaling and hepcidin expression induced by HJV in Hep3B and Huh-7 cells. (A) Total RNA from Hep3B and Huh-7 cells and human liver was extracted for RT-PCR to determine the expression of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6, and BMP-9. Purified plasmid cDNAs containing these ligands were used as positive controls. (B-E) Hep3B cells (B,C) or Huh-7 cells (D,E) were transfected with the BMP-responsive firefly luciferase reporter (BRE-Luc) and pRL-TK Renilla luciferase vector, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA or siRNA specific for BMP-2, BMP-4, BMP-6, or BMP-7 (Control, si-BMP-2, siBMP-4, siBMP-6, and si-BMP-7; 60 nM) for 46 hours prior to measurement of luciferase activity. Firefly luciferase values were normalized for transfection efficiency relative to Renilla activity. Values shown are the means of triplicate measurements plus or minus SD. Basal BRE luciferase activities in the absence or presence of BMP-2, BMP-4, BMP-6, and BMP-7 siRNAs (dotted square in panels B and D) were replotted in panels C and E. (F) Hep3B cells were transfected with the hepcidin promoter firefly luciferase reporter (Hep-Luc) and pRL-TK Renilla luciferase vector, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA or si-BMP-2, si-BMP-4, si-BMP-6, or si-BMP-7 at 60 nM. After 46 hours, cell lysates were analyzed for luciferase activity as in panels B-E. (G,H) Hep3B cells (G) or Huh-7 cells (H) were transfected with empty vector (pcDNA3) and control siRNA or with Flag-HJV cDNA in combination with control siRNA, si-BMP-2, si-BMP-4, si-BMP-6, or si-BMP-7 at 60 nM. After 46 hours, cells were collected for RNA extraction and real time RT-PCR analyses to quantify hepcidin and RPL19 mRNA levels. Hepcidin expression values were normalized to RPL19 mRNA levels. Values shown are the means of triplicate measurements plus or minus SD. *P < .05; **P < .01; ***P < .001.

Impact of siRNA-mediated specific inhibition of BMP ligand expression on BMP signaling and hepcidin expression induced by HJV in Hep3B and Huh-7 cells. (A) Total RNA from Hep3B and Huh-7 cells and human liver was extracted for RT-PCR to determine the expression of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6, and BMP-9. Purified plasmid cDNAs containing these ligands were used as positive controls. (B-E) Hep3B cells (B,C) or Huh-7 cells (D,E) were transfected with the BMP-responsive firefly luciferase reporter (BRE-Luc) and pRL-TK Renilla luciferase vector, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA or siRNA specific for BMP-2, BMP-4, BMP-6, or BMP-7 (Control, si-BMP-2, siBMP-4, siBMP-6, and si-BMP-7; 60 nM) for 46 hours prior to measurement of luciferase activity. Firefly luciferase values were normalized for transfection efficiency relative to Renilla activity. Values shown are the means of triplicate measurements plus or minus SD. Basal BRE luciferase activities in the absence or presence of BMP-2, BMP-4, BMP-6, and BMP-7 siRNAs (dotted square in panels B and D) were replotted in panels C and E. (F) Hep3B cells were transfected with the hepcidin promoter firefly luciferase reporter (Hep-Luc) and pRL-TK Renilla luciferase vector, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA or si-BMP-2, si-BMP-4, si-BMP-6, or si-BMP-7 at 60 nM. After 46 hours, cell lysates were analyzed for luciferase activity as in panels B-E. (G,H) Hep3B cells (G) or Huh-7 cells (H) were transfected with empty vector (pcDNA3) and control siRNA or with Flag-HJV cDNA in combination with control siRNA, si-BMP-2, si-BMP-4, si-BMP-6, or si-BMP-7 at 60 nM. After 46 hours, cells were collected for RNA extraction and real time RT-PCR analyses to quantify hepcidin and RPL19 mRNA levels. Hepcidin expression values were normalized to RPL19 mRNA levels. Values shown are the means of triplicate measurements plus or minus SD. *P < .05; **P < .01; ***P < .001.

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