Were approved by the University of Perugia’s Animal Care Committee

Were approved by the University of Perugia’s Animal Care Committee JNJ-42756493 according to the Italian guideline for care and use of laboratory animals. The latest ID for this project is #98/ 2010-B. The authorization was released to Prof. Stefano Fiorucci, as a principal investigator, on May 19, 2010. C57BL/6 male mice were from Harlan Laboratories, while the colony of C57BL/6jMucosal damage scoreMucosal damage scoring was analyzed considering the presence of indurations, edema, thickness and evidence of mucosal hemorrhage, as indicated previously [19]. For histologic examination, a section of the distal colon from each animal was fixed inFXR Is a Novel TLR-9 Target GeneFigure 6. A conserved IRF7 responsive element is expressed in the promoter of FXR. (A) order Erastin analysis of the FXR promoter was performed with the on-line software TFsearch. The human FXR 5’flanking region contains an IRF7-RE at 2602 base pairs with respect to the transcriptional start site ATG. The murine FXR 5’flanking region contains an IRF7-RE at 2787 base pairs with respect to the transcriptional start site ATG. (B) Quantitative RTPCR of FXR and IRF7 genes was carried out on RNA purified from Raw264.7 cells stimulated with CpG. Data are mean 6 SE of of 4 experiments. *P,0.05 versus not treated cells. (C) Western Blotting analysis of FXR, IRF7 and tubulin was performed on protein extracts from Raw264.7 cells stimulated with CpG. The image shown is one of three showing the same pattern. (D) Densitometric analysis of Western blot bands carried out using the Image J software. Data are the mean of three experiments. (E) Transactivation of IRF7-RE. Three copies of the murine IRF7-RE were cloned into the luciferase reporter vector pGL4. Raw264.7 cells were transiently transfected with this construct and forty-eight hours post-transfection cells were stimulated with increasing concentrations of CpG. Cellular extracts were subsequently assayed for luciferase activity. Data are the mean 6 S.E. of 3 experiments carried out in triplicate. *P,0.05 versus not treated cells. doi:10.1371/journal.pone.0054472.g10 formalin, embedded in paraffin, sectioned, and stained with Haematoxylin and Eosin (H E).Western BlottingTotal proteins from Raw264.7 cells were separated by SDS PAGE, transferred to nitrocellulose membranes (Bio-Rad) and probed with primary antibodies FXR (Abcam), IRF-7 (SantaCruz) and a-tubulin (Sigma). The anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) was used as the secondary antibody and specific protein bands were visualized using the Super Signal West Dura reagent (Pierce), following the manufacturer’s suggested protocol. Films were scanned and the densitometry analysis performed using the Image J software (NIH, Bethesda, MD, USA).Cell cultureThe Raw264.7, a mouse macrophage cell line, was purchased from the ATCC-LGC Standards and cultured in D-MEM supplemented with 10 Fetal Bovine serum, 1 L-glutamine and 1 penicillin/streptomycin. Serum starved Raw264.7 cells were stimulated for 18 hours with CpG ODN 2395 (2 mg/ml) in D-MEM. After stimulation cells were divided in two aliquots: 56106 cells were lysed in 1 ml TRIZOL reagent (to assess the relative mRNA expression of FXR and IRF-7 by quantitative Real-Time PCR) while 56106 cells were lysed in 500 ml SDS lysis buffer containing b-mercaptoethanol (to measure protein expression of FXR and IRF-7 by Western blotting).Isolation and culture of human and mouse monocytesHuman CD14 derived Peripheral blood mononuclear ce.Were approved by the University of Perugia’s Animal Care Committee according to the Italian guideline for care and use of laboratory animals. The latest ID for this project is #98/ 2010-B. The authorization was released to Prof. Stefano Fiorucci, as a principal investigator, on May 19, 2010. C57BL/6 male mice were from Harlan Laboratories, while the colony of C57BL/6jMucosal damage scoreMucosal damage scoring was analyzed considering the presence of indurations, edema, thickness and evidence of mucosal hemorrhage, as indicated previously [19]. For histologic examination, a section of the distal colon from each animal was fixed inFXR Is a Novel TLR-9 Target GeneFigure 6. A conserved IRF7 responsive element is expressed in the promoter of FXR. (A) Analysis of the FXR promoter was performed with the on-line software TFsearch. The human FXR 5’flanking region contains an IRF7-RE at 2602 base pairs with respect to the transcriptional start site ATG. The murine FXR 5’flanking region contains an IRF7-RE at 2787 base pairs with respect to the transcriptional start site ATG. (B) Quantitative RTPCR of FXR and IRF7 genes was carried out on RNA purified from Raw264.7 cells stimulated with CpG. Data are mean 6 SE of of 4 experiments. *P,0.05 versus not treated cells. (C) Western Blotting analysis of FXR, IRF7 and tubulin was performed on protein extracts from Raw264.7 cells stimulated with CpG. The image shown is one of three showing the same pattern. (D) Densitometric analysis of Western blot bands carried out using the Image J software. Data are the mean of three experiments. (E) Transactivation of IRF7-RE. Three copies of the murine IRF7-RE were cloned into the luciferase reporter vector pGL4. Raw264.7 cells were transiently transfected with this construct and forty-eight hours post-transfection cells were stimulated with increasing concentrations of CpG. Cellular extracts were subsequently assayed for luciferase activity. Data are the mean 6 S.E. of 3 experiments carried out in triplicate. *P,0.05 versus not treated cells. doi:10.1371/journal.pone.0054472.g10 formalin, embedded in paraffin, sectioned, and stained with Haematoxylin and Eosin (H E).Western BlottingTotal proteins from Raw264.7 cells were separated by SDS PAGE, transferred to nitrocellulose membranes (Bio-Rad) and probed with primary antibodies FXR (Abcam), IRF-7 (SantaCruz) and a-tubulin (Sigma). The anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) was used as the secondary antibody and specific protein bands were visualized using the Super Signal West Dura reagent (Pierce), following the manufacturer’s suggested protocol. Films were scanned and the densitometry analysis performed using the Image J software (NIH, Bethesda, MD, USA).Cell cultureThe Raw264.7, a mouse macrophage cell line, was purchased from the ATCC-LGC Standards and cultured in D-MEM supplemented with 10 Fetal Bovine serum, 1 L-glutamine and 1 penicillin/streptomycin. Serum starved Raw264.7 cells were stimulated for 18 hours with CpG ODN 2395 (2 mg/ml) in D-MEM. After stimulation cells were divided in two aliquots: 56106 cells were lysed in 1 ml TRIZOL reagent (to assess the relative mRNA expression of FXR and IRF-7 by quantitative Real-Time PCR) while 56106 cells were lysed in 500 ml SDS lysis buffer containing b-mercaptoethanol (to measure protein expression of FXR and IRF-7 by Western blotting).Isolation and culture of human and mouse monocytesHuman CD14 derived Peripheral blood mononuclear ce.