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Blood, Vol. 111, Issue 10, 5195-5204, May 15, 2008
Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin
Blood Xia et al.
111: 5195
Supplemental materials for: Xia et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 60.1 KB)
- Table S1. Sequences, expected product sizes, and GenBank accession numbers for the primers used in RT-PCR (PDF, 18.1 KB) -
Only primers that have not been published elsewhere are shown.
- Table S2. Sequences and GenBank accession numbers of siRNAs for human BMP ligands, type II receptors and mouse RGMa (PDF, 13.9 KB) -
Only siRNA sequences that have not been published elsewhere are shown.
- Table S3. Sequences, expected product sizes, and GenBank accession numbers for the primers used in real time PCR (PDF, 16.2 KB)
- Figure S1. Specificity and efficacy of siRNA targeting human BMP-2, BMP-4, BMP-6 and BMP-7 expression in Hep3B cells (JPG, 78.9 KB)
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siRNA (60 nM) derived from sequences of BMP-2, BMP-4 BMP-6 and BMP-7 were employed to decrease their expression in Hep3B cells. mRNA levels for BMP-2 (A), BMP-4 (B), BMP-6 (C) and BMP-7 (D) were measured 46 h after cells were transfected with specific siRNA by quantitative real time PCR, were normalized to RPL19 mRNA levels, and are expressed as a fraction of values from cells treated with control negative siRNA. Values shown are the mean of triplicate measurements ± S.E. BMP2-, BMP4-, BMP6- and BMP7-specific siRNA reduced the expression of those genes by 97%, 90%, 90% and 80% respectively. ***, p < 0.001.
- Figure S2. Impact of anti-BMP-6 antibody treatment on basal and HJV-mediated BMP signaling in Huh-7 and Hep3B cells (JPG, 60.3 KB)
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Hep3B (A) or Huh-7 (B) cells were transfected with BRE-Luc and pRL-TK, either alone or with increasing amounts of Flag-HJV cDNA, in the absence (control) or presence of 15 µg/ml of anti-BMP-6 antibody for 46 h prior to measurement of luciferase activity. Luciferase values were normalized for transfection efficiency relative to Renilla activity. Values shown are the mean of triplicate measurements ± S.D. *, p < 0.05; ***, p < 0.001.
- Figure S3. BMPRII and ALK6 are not expressed in human liver (JPG, 29.7 KB)
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Previously described real-time PCR primers26 for BMPRII and real-time PCR primers described in Table S3 for ALK6 were used to determine BMPRII and ALK6 expression in human liver with KGN cells as a positive control and water as a negative control.
- Figure S4. Specificity and efficacy of siRNA targeting human BMPRII, ActRIIA and ActRIIB expression in Hep3B cells (JPG, 74.6 KB)
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(A) siRNA (40 nM) derived from sequences of BMPRII, ActRIIA and ActRIIB were employed to decrease the mRNA expression of endogenous BMP type II receptors in Hep3B cells. mRNA levels were measured as in Figure S1. BMPRII-, ActRIIA- and ActRIIB-specific siRNA reduced the expression of those genes by 80-90%. ***, p < 0.001. (B) siRNAs to BMPRII, ActRIIA and ActRIIB were employed to decrease the protein expression of transfected BMP type II receptors. Hep3B cells were transfected with BMPRII-GFP, ActRIIA-Myc or ActRIIB-CFP plasmid with or without corresponding siRNA. The protein levels were measured by Western blotting using anti-GFP ( -GFP) or anti-Myc (-Myc). Two repeats of control and receptor siRNA transfections were presented. Actin staining (-actin) was used as loading control.
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