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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:
- Figure S5. Impact of siRNA mediated specific inhibition of BMP type II receptor expression on BMP signaling induced by HJV in Huh-7 and HepG2 cells (JPG, 89.4 KB)
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(A) Expression of BMP type II recpetors BMPRII, ActRIIA and ActRIIB in Huh-7 and HepG2 cells (RT-PCR). (B and D) Impact of siRNA targeting of BMPRII, ActRIIA and ActRIIB on BMP-2 signaling in Huh-7 (B) or HepG2 (D) cells. Cells were transfected with the BMP-responsive firefly luciferase reporter (BRE-Luc) and pRL-TK Renilla luciferase vector, in combination with control siRNA or siRNA specific for BMPRII, ActRIIA or ActRIIB (40 nM). Transfected cells were then incubated in the absence (bar 1) or presence (bars 2-5) of 20 ng/ml BMP-2. Luciferase activity was measured as in Figure S2. (C and E) Impact of siRNA targeting of BMPRII, ActRIIA and ActRIIB on HJV-mediated BMP signaling in Huh-7 (C) or HepG2 (E) cells. Cells were transfected with BRE-Luc and pRL-TK, in combination with control siRNA or siRNA specific for BMPRII, ActRIIA or ActRIIB (60 nM), in the absence (bar 1) or presence (bars 2-5) of HJV cDNA (8ng/ml) for 46 h prior to measurement of luciferase activity. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
- Figure S6. BMP-2 and BMP-4 are the sole ligands used by the co-receptor HJV to induce BMP signlaing in KGN cells (JPG, 79.7 KB)
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(A) Impact of siRNA targeting of BMP-2, BMP-4 and BMP6 on basal and HJV mediated BMP signaling. KGN cells were transfected with BRE-Luc and pRL-TK, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA, si-BMP2, si-BMP4 or si-BMP6 (60 nM) for 46 h prior to measurement of luciferase activity. HJV-induced BMP signaling was reduced by siRNA inhibition of BMP-2 or BMP-4 expression but not by inhibition of BMP-6 expression. (B) Impact of siRNA targeting of BMP-2, BMP-4 and BMP-6 on basal BMP signaling (replotted from the dotted square in A). Basal BMP signaling was reduced by inhibition of BMP-2 or BMP-4 expression but not by inhibition of BMP-6 expression indicating that BMP-6 does not contribute to endogenous BMP signaling or HJV-mediated BMP signaling. (C) Impact of anti-BMP-6 antibody treatment on basal BMP signaling in KGN and Huh-7 cells. KGN cells or Huh-7 cells were transfected with BRE-Luc and pRL-TK in the absence (control) or presence of 15 µg/ml of BMP-6 antibody. Basal BMP signaling was not altered by specific anti-BMP-6 antibody in KGN cells. (D) Impact of combined BMP2 and BMP4 siRNAs on HJV mediated BMP signaling. KGN cells were transfected with BRE-Luc and pRL-TK, either alone or with increasing amounts of Flag-HJV cDNA, in combination with control siRNA, si-BMP2, si-BMP4 or si-BMP2 combined with si-BMP4 (40 nM each). Control siRNA was used to equalize the total amount of siRNA. HJV-induced BMP signaling was abolished to baseline by co-transfection of both BMP-2 and BMP-4 siRNAs. *, p < 0.05; **, p < 0.01.
- Figure S7. Impact of siRNA targeting of BMPRII, ActRIIA and ActRIIB on HJV-mediated BMP signaling in KGN cells in the absence (A) or presence (B) of anti-BMP-6 antibody (JPG, 53.5 KB)
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KGN cells were transfected with BRE-Luc and pRL-TK, in combination with control siRNA or siRNA specific for BMPRII, ActRIIA or ActRIIB (40 nM), in the absence (bar 1) or presence (bars 2-5) of HJV cDNA (8ng/ml), with (B) or without (A) 15 µg/ml of BMP-6 antibody for 46 h prior to measurement of luciferase activity. **, p < 0.01.
- Figure S8. Specificity and efficacy of siRNA targeting human ALK2, ALK3 and ALK6 expression in Hep3B cells (JPG, 70.9 KB)
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(A) siRNA (60 nM) derived from sequences of ALK2, ALK3 and ALK6 were employed to decrease the mRNA expression of endogenous BMP type I receptors in Hep3B cells. mRNA levels were measured measured as in Figure S1. ALK2-, ALK3- and ALK6- specific siRNA reduced the expression of those genes by 75-90%. ***, p < 0.001. (B) siRNAs to ALK2, ALK3 and ALK6 were employed to decrease the protein expression of transfected BMP type I receptors. Hep3B cells were transfected with HA-ALK2, ALK3 or HA-ALK6 plasmid with or without corresponding siRNA. The protein levels were measured by Western blotting using anti-HA ( -HA) or anti-ALK3 (-ALK3). Two repeats of control and receptor siRNA transfections were presented. Actin staining (-actin) was used as loading control.
- Figure S9. Impact of siRNA targeting of ALK2 and ALK3 on HJV-mediated BMP signaling in Huh-7 cells in the absence (A) or presence (B) of BMP-6 antibody (JPG, 56.5 KB)
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Huh-7 cells were transfected with BRE-Luc and pRL-TK, in combination with control siRNA or siRNA specific for ALK2, or ALK3, in the absence (bar 1) or presence (bars 2-4) of HJV cDNA, with (B) or without (A) 15 mg/ml of BMP-6 antibody for 46 h prior to measurement of luciferase activity. **, p < 0.01; ***, p < 0.001.
- Figure S10. Co-immunoprecipitation of neogenin and HJV (JPG, 52.6 KB)
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(A) Expression of HA-Neogenin in HEK293 and Hep3B cells. Empty pcDNA3 or HA-Neogenin (100 ng/ml) was transfected into HEK293 or Hep3B cells in 10 cm dishes using Effectene. Approximately 46 hrs later, whole lysates were collected in 500 µl Tris-HCl buffer containing 10 mM Tris-HCl, pH7.4, 150 mM NaCl, 1% Triton-100, and protease inhibitors. Cell lysates were subjected to reducing SDS-PAGE and immunoblotting with anti-neogenin or anti-HA. The arrowheads indicate specific neogenin bands. (B) Co-immunoprecipitation of HA-Neogenin and Flag-HJV. HA-Neogenin (1.5 µg), Flag-HJV (0.5 µg) or both were transfected into HEK293 cells in 10 cm dishes using Effectene. Cell lysates were immunoprecipitated with ANTI-FLAG M2 Affinity Gel. The lysates and immunoprecipitates were subjected to SDS-PAGE under reducing conditions, and transferred to PVDF membrane. Anti-HA antibody was used to detect HA-Neogenin in cell lysates and immunoprecipitates. The membranes were stripped and re-probed with Flag antibody M5.
- Figure S11. Efficacy of siRNA targeting human neogenin expression in Hep3B cells (JPG, 58.9 KB)
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A mixture of three neogenin siRNA sequences (Table S2) was employed to decrease neogenin expression in Hep3B cells. mRNA levels (upper panel) were measured 46 h after transfection by quantitative real time PCR. Endogenous neogenin mRNA expression was reduced by 75% with neogenin siRNA in Hep3B cells. Neogenin protein levels (lower panel) were also measured by Western blotting using anti-Neogenin (-NEN). Two repeats of control and neogenin siRNA transfections were presented. Actin staining (-actin) was used as loading control. ***, p < 0.001.
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