Journal: RNA

Article Title: MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming

doi: 10.1261/rna.043745.113

Figure Lengend Snippet: Wisp1 is a key regulator of extracellular matrix genes. ( A ) Wisp1 regulates expression of several ECM genes. Expression of Tgfbi, Igfbp5, Dkk2, Nov, and Ccl20 were dramatically changed upon Wisp1 knockdown. Uninfected and 4F-infected MEFs were transfected with siWisp1 for 2 d and total RNAs were harvested for RT-qPCR analysis of different ECM genes. Error bars represent two independent experiments with duplicate wells. ( B ) Knockdown of Nov, Dkk2, and Tgfbi significantly enhances iPSC generation. MEFs were transduced with 4F at Day 0 and transfected with siRNAs at Day 5 post-infection. GFP + colonies were quantified at around Days 11–13. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( C ) Overexpression of Wisp1-regulated ECM genes compromises reprogramming. The indicated ECM genes were cloned into pMX retroviral vectors. MEFs were transduced with 4F plus the indicated ECM genes and GFP + colonies were quantified at around Days 11–13. Data were normalized to pMX-RFP-transduced cells. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( D ) Addition of recombinant ECM proteins compromises reprogramming. Purified recombinant TGFBI, DKK2, NOV, and CCL20 were added at a final concentration of 100 ng/mL to cultures of 4F-MEFs undergoing reprogramming. GFP + colonies were quantified at Days 11–13. Error bars represent two independent experiments with triplicate wells. (*) P < 0.05.

Article Snippet: Recombinant proteins were obtained from commercial sources as follows: mouse Dkk2 (R&D systems, 2435DK/CF), human NOV/CCN3 (R&D systems, 1640NV), human TGFBI (Prospec, #PRO-568), CCL20 (R&D systems, 760-M3).

Techniques: Expressing, Knockdown, Infection, Transfection, Quantitative RT-PCR, Transduction, Over Expression, Clone Assay, Retroviral, Recombinant, Purification, Concentration Assay

Journal: Mucosal Immunology

Article Title: TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

doi: 10.1038/mi.2013.5

Figure Lengend Snippet: CCL20 is secreted in a Toll-like receptor 1 (TLR1)-dependent manner from intestinal cells. Levels of CCL20 protein ( a ) and messenger RNA (mRNA; b ) from various mucosal tissues 3 days after oral Y. enterocolitica infection. Data are pooled from two independent experiments ( n =4–6 mice per group). ( c ) Levels of CCL20 mRNA in intestinal epithelial cells (IECs; CD13+CD45-) and lamina propria immune cells (LPCs; CD13-CD45+) sorted from wild-type (WT) and TLR1 −/− mice. Purified cells were stimulated with TLR2/1 ligand, TLR2/6 ligand, and Y. enterocolitica (Ye). Data are the average from three to four individual mice. ( d ) Level of CCL20 from Caco-2 cells transfected with control vector (control), WT TLR1 (I602I), or TLR1 containing a single-nucleotide polymorphism (I602S) 18 h after stimulation with TLR ligands or Y. enterocolitica lysate (Ye). Data are pooled from three independent experiments. ( n =6). * P <0.05, ** P <0.01, *** P <0.001. Student's unpaired t -test. MLN, mesenteric lymph node.

Article Snippet: Custom-made goat anti-murine CCL20 was prepared by Invitrogen Life Technologies (Grand Island, NY) by multiple site immunization of a goat with recombinant mouse CCL20 (R&D System, Minneapolis, MN) emulsified in Complete Freund's Adjuvant.

Techniques: Infection, Purification, Transfection, Control, Plasmid Preparation

Journal: Mucosal Immunology

Article Title: TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

doi: 10.1038/mi.2013.5

Figure Lengend Snippet: CCL20 production is dependent upon Toll-like receptor 1 (TLR1) signaling and not invasion or type III secretion system (T3SS). ( a ) Number of migrated cells toward supernatants collected from Caco-2 epithelial cells transfected with wild-type (WT) TLR1 (I602I) and stimulated with wild-type Y. enterocolitica (8081), Y. enterocolitica lacking LcrV (Δ lcrV ), T3SS (Δ ysc ), or invasin (Δ invA ). Recombinant human CCL20 (rCCL20) and neutralization of CCL20 (aCCL20) were used as controls. Data are the mean±s.e.m. of pooled from three independent experiments. * P <0.01, ** P <0.001 (Student's unpaired t -test). ( b ) In vivo production of CCL20 from IECs of TLR1 −/− or wild-type littermate control mice infected with 1 × 10 6 Y. enterocolitica or mutants after 72 h. Data are the mean±s.e.m. of six individual mice pooled from two independent experiments. * P <0.01, ** P <0.001 (Student's unpaired t -test).

Article Snippet: Custom-made goat anti-murine CCL20 was prepared by Invitrogen Life Technologies (Grand Island, NY) by multiple site immunization of a goat with recombinant mouse CCL20 (R&D System, Minneapolis, MN) emulsified in Complete Freund's Adjuvant.

Techniques: Transfection, Recombinant, Neutralization, In Vivo, Control, Infection

Journal: Mucosal Immunology

Article Title: TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

doi: 10.1038/mi.2013.5

Figure Lengend Snippet: CCL20 is important for survival and clearance of Y. enterocolitica . ( a ) Survival of mice fed 1 × 10 5 c.f.u. (colony-forming unit) Y. enterocolitica and treated with anti-CCL2 or anti-IgG polyclonal serum every other day for 10 days. N =10 mice per group; * P =0.0132 (Wilcoxon Log-Rank test). ( b ) Bacterial burden in the mesenteric lymph node 3 days post infection. Data are pooled from two independent experiments ( n =5 mice per group). ** P <0.01 (Student's paired t -test).

Article Snippet: Custom-made goat anti-murine CCL20 was prepared by Invitrogen Life Technologies (Grand Island, NY) by multiple site immunization of a goat with recombinant mouse CCL20 (R&D System, Minneapolis, MN) emulsified in Complete Freund's Adjuvant.

Techniques: Infection

Journal: Mucosal Immunology

Article Title: TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

doi: 10.1038/mi.2013.5

Figure Lengend Snippet: Toll-like receptor 1 (TLR1) and CCL20 are important for recruitment of CCR6+ CD11c+ cells to the Peyer's patch (PP) during oral Y. enterocolitica infection. Flow cytometric analysis of dendritic cell (DC) population in PP of anti-CCL20-treated ( a ) and TLR1 −/− ( b ) mice. Data are representative of five individual mice pooled from two experiments. ( c ) Total cell number of CCR6+CD11c+ cells. ( d ) Bone marrow chimera's were performed by reconstituting TLR1 −/− mice with wild-type (WT) hematopoietic cells (WT→TLR1 −/− ) and by reconstituting WT mice with TLR1 −/− hematopoietic cells (TLR1 −/− →WT). CCR6+ DC in the PP of the bone marrow chimera's were identified by flow cytometric analysis (left) and total cell count (right) 3 days after infection with Y. enterocolitica . Data are pooled from two independent experiments ( n =3–6 mice per group). * P <0.05, ** P <0.01, *** P <0.001 (Student's unpaired t -test).

Article Snippet: Custom-made goat anti-murine CCL20 was prepared by Invitrogen Life Technologies (Grand Island, NY) by multiple site immunization of a goat with recombinant mouse CCL20 (R&D System, Minneapolis, MN) emulsified in Complete Freund's Adjuvant.

Techniques: Infection, Cell Counting

Journal: Mucosal Immunology

Article Title: TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

doi: 10.1038/mi.2013.5

Figure Lengend Snippet: Absence of CCL20 impacts interleukin (IL)-17 priming and IgA secretion. ( a ) Levels of IL-23, IL-6, and IL-10 in the Peyer's patch of TLR1 −/− or anti-CCL20-treated mice infected orally with Y. enterocolitica . Data are the mean±s.e.m. pooled from two independent experiments ( n =6). ( b ) Levels of IL-17 from isolated LP CD4 T cells re-stimulated with irradiated antigen-presenting cell and Y. enterocolitica lysate. Data are the mean±s.e.m. pooled from two independent experiments ( n =6). ( c ) Fecal anti- Yersinia IgA levels from mice collected at day 14 post infection. Data are pooled from two independent experiments ( n =6 mice per group). ( d ) Messenger RNA (mRNA) levels of IL-6 and IL-23 from CCR6+ and CCR6- CD11c+ sorted Peyer's patch cells 3 days post infection. Fold induction is compared with uninfected controls. Data are pooled from two independent experiments ( n =5–6 mice per group). ( e ) Purified CCR6+ and CCR6- dendritic cells (DCs) were sorted from wild-type mice infected with Y. enterocolitica . The purified DCs were co-cultured with CD4+ ovalbumin-specific T cells (OT2) and ovalbumin. Supernatants were analyzed after 3 days for levels of IL-17 (left). CD4 T cells were sorted from the co-cultures and mRNA level of the transcription factor for rorgt was determined. ( f ) Levels of IL-17 in co-cultures similar to those in 5e with the addition of neutralizing antibodies to IL-6 and IL-23. Data are the average±s.e.m. pooled from three independent experiments. * P <0.01, ** P <0.01 (Student's unpaired t -test).

Article Snippet: Custom-made goat anti-murine CCL20 was prepared by Invitrogen Life Technologies (Grand Island, NY) by multiple site immunization of a goat with recombinant mouse CCL20 (R&D System, Minneapolis, MN) emulsified in Complete Freund's Adjuvant.

Techniques: Infection, Isolation, Irradiation, Purification, Cell Culture