Impact of siRNA-mediated specific inhibition of BMP type I receptor expression on BMP signaling and hepcidin expression induced by HJV in Hep3B, KGN, and Huh-7 cells. (A) Expression of BMP type I receptors ALK2, ALK3, and ALK6 in Hep3B, Huh-7, and KGN cells, and human liver (RT-PCR). (B-I) Hep3B (B-D), KGN (E,F), or Huh-7 cells (G-I) were transfected with BRE-Luc or CAGA-Luc and pRL-TK Renilla luciferase vector, in combination with control siRNA or siRNA specific for ALK2, ALK3, and ALK6 (60 nM). Cells were incubated in the absence or presence of 20 ng/mL BMP-2 (B,E,G), 20 ng/mL activin A (D), or 20 ng/mL BMP-6 (H), or after cotransfection with 8 ng/mL HJV cDNA (C,F,I). Cell lysates were analyzed for luciferase activity as in Figure 1B-E. (J,K) Hep3B (J) or Huh-7 cells (K) were transfected with Hep-Luc and pRL-TK, in combination with control siRNA or siRNA specific for ALK2, ALK3, or ALK6 (60 nM), in the absence or presence of HJV cDNA (8 ng/mL) for 46 hours prior to measurement of luciferase activity. Values shown are the means of triplicate measurements plus or minus SD. *P < .05; **P < .01; ***P < .001.