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.