Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: Identification of TLR6 as a novel regulator of ferroportin protein. (A) A stable and doxycycline-inducible HeLa cell line expressing a human ferroportin-Renilla luciferase fusion protein (Fpn-RLuc) was used for RNAi screening. Renilla luciferase activity (Rluc), used as a reporter of ferroportin expression, was measured 70 hours after reverse transfection of siRNA pools. The screen was performed in duplicates and the cellHTS2 software was used for data analysis. (B) Rluc activity was measured upon scramble (scr) or TLR6 interference with pooled siRNAs in the HeLa cell line expressing Fpn-Rluc and in a HeLa cell line expressing only the reporter protein. Data are presented as means ± SEM from at least 4 independent experiments. *P < .05; Student t test. (C,F) Western blot analysis of endogenous ferroportin expression in BMDMs isolated from WT or TLR6-deficient (TLR6 KO) mice or TLR2-deficient (TLR2 KO) mice; β-actin was used as loading control. Western blot images were acquired and quantified with the Vilber Lourmat Fusion-FX Chemiluminescence system. (D-E) Ferroportin and hepcidin mRNA levels were determined by qRT-PCR and calibrated to 36B4 mRNA levels. Data are means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. **P < .01; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Software, Western Blot, Isolation, Quantitative RT-PCR, Derivative Assay

Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: FSL1-mediated TLR2/6 ligation reduces ferroportin expression in BMDMs without activating hepcidin mRNA expression. (A,C) qRT-PCR analysis of ferroportin mRNA in BMDMs from WT and TLR6-deficient mice (A), and from WT and TLR2-deficient mice (C) stimulated with FSL1 (20 ng/mL or 100 ng/mL) for the indicated time. (B,D) Western blot analysis and quantification of ferroportin expression in BMDMs from WT and TLR6-deficient mice (B) and from WT and TLR2-deficient mice (D) treated with 100 ng/mL FSL1 for 24 hours. β-actin detection ascertains equal sample loading. (E-F) Ferroportin and hepcidin mRNA expression in BMDMs after FSL1 and LPS (100 ng/mL) stimulation. mRNA levels were normalized to 36B4 mRNA levels. All data are reported as means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. *P < .05; **P < .01; ***P < .001; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Ligation, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: Ferroportin downregulation is mediated by TLR2 and TLR4 ligands whereas hepcidin activation is limited to the TLR4 ligand LPS in BMDMs. (A,C-D) Ferroportin mRNA expression was determined by qRT-PCR in WT (A), TLR6-deficient (C), and TLR2-deficient (D) BMDMs stimulated with 100 ng/mL TLR2 ligands (FSL1, PAM3CSK4, PamOct2C-(VPG)4VPGKG) or TLR4 ligand (LPS) for the indicated time. (B) Western blot analysis and quantification of ferroportin expression in BMDMs from WT mice treated with 100 ng/mL LPS and PAM3CSK4 for 24 hours. β-actin was used as loading control. (E-G) Hepcidin mRNA expression was analyzed in the same samples. The mRNA quantification was calibrated to 36B4 mRNA levels. All data are reported as means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. *P < .05; **P < .01; ***P < .001; ****P < .0001; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay