Journal: Scientific Reports

Article Title: Inhibition of intimal hyperplasia in murine aortic allografts by administration of a small-molecule TLR4 inhibitor TAK-242

doi: 10.1038/s41598-017-16160-4

Figure Lengend Snippet: TAK-242 downregulates proinflammatory cytokines in grafts and NF-κB signaling pathway in graft macrophages. TAK-242 treatment significantly reduced expression of ( A ) IL-12 (P = 0.0491), ( B ) IL-6 (P = 0.012), TNFα (P = 0.016), iNOS (P = 0.005), IL-1β (P = 0.043), and ( C ) CCL2 (P < 0.001) and increased expression of (A) IL-4 (P < 0.001) in allografts as compared with vehicle treatment. There was no significant difference in the expression of other genes ( A , B , C , D ) between the two groups. ( E ) TAK-242 treatment significantly decreased TRAF6 (P = 0.036) and CCL2 (P = 0.023) expression and decreased phosphorylation of IκBα (P = 0.010) and p65 (P = 0.049) in graft macrophages, as compared with vehicle treatment. (n = 4 mice for per group *P < 0.05, **P < 0.05, ***P < 0.001).

Article Snippet: Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber.

Techniques: Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Inhibition of intimal hyperplasia in murine aortic allografts by administration of a small-molecule TLR4 inhibitor TAK-242

doi: 10.1038/s41598-017-16160-4

Figure Lengend Snippet: TAK-242 reduces the migration of VSMCs towards CCL2. ( A ) VSMCs migration was induced by macrophages and neutralized by anti-CCL2 antibody. The VSMC migration towards CCL2 was examined by transwell assay at 10 ( B) and 24 hours ( C ) after seeding. At 10 hours, CCL2 induced VSMC migration in a dose-dependent manner, irrespective of the presence of FBS. At 24 hours, 10 ng/ml CCL2 significantly promoted VSMC migration. There was no significant difference in the number of migrated VSMCs between the high (100 ng/ml) and low (10 ng/ml) concentrations of CCL2. (n = 5 independent experiments, # Bonferroni corrected level of significance <0.05).

Article Snippet: Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber.

Techniques: Migration, Transwell Assay

Journal: Scientific Reports

Article Title: Inhibition of intimal hyperplasia in murine aortic allografts by administration of a small-molecule TLR4 inhibitor TAK-242

doi: 10.1038/s41598-017-16160-4

Figure Lengend Snippet: The sequences of the primers used.

Article Snippet: Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber.

Techniques:

Journal: eLife

Article Title: CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

doi: 10.7554/eLife.80443

Figure Lengend Snippet: Transwell assays were used to quantify chemotaxis of thymocyte subsets to the indicated concentrations of the CCR4 ligands CCL17 ( A ) and CCL22 ( B ) and the CCR7 ligands CCL19 ( C ) and CCL21 ( D ). Migration index was calculated as the frequency of input cells of each subset that migrated toward the chemokine relative to the frequency of input cells that migrated in the absence of chemokine (Blank). Data were compiled from three independent experiments with triplicate wells per assay. Two-way ANOVA with Dunnett’s multiple comparisons correction was used for statistical analysis (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: peptide, recombinant protein , Recombinant Murine CCL21 , Peprotech , Cat # 250–13 , .

Techniques: Chemotaxis Assay, Migration

Journal: eLife

Article Title: CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

doi: 10.7554/eLife.80443

Figure Lengend Snippet: ( A ) Representative images of H&E stains from the colon of 5- to 6-month-old WT, Ccr4 −/− , Ccr7 −/− , or DKO mice (left). Bars, 200 µm. Histological lesion score was quantified by a pathologist blinded to genotype (right). One-way ANOVA was used to determine significance between groups (mean ± SEM; ***p < 0.001). ( B ) Representative images of H&E stains from extraorbital lacrimal glands of 5- to 6-month-old WT, Ccr4 −/− , Ccr7 −/− , or DKO mice (left). Bars, 500 µm. Arrows indicate lymphocytic infiltrate foci. The number of infiltrate foci per 10 mm 2 was quantified (right), and one-way ANOVA was used to test significance between groups (mean ± SEM; *p < 0.05, ***p > 0.001). ( C ) Representative immunofluorescent images of CD4 (green), CD8 (red), and B220 (blue) on cryosections of inguinal lymph nodes from 5- to 6-month-old WT and Ccr4 −/− mice (left). Frequency of moderate lymphoid hyperplasia in mLNs and spleens from 12- to 13-month-old WT, Ccr4 −/− , and Ccr7 −/− mice, as determined by a veterinary pathologist, blinded to genotype (right). Fisher’s exact test was used to determine significance. Bars, 300µm. Expression of CCR4 ligands ( D ) and CCR7 ligands ( E ) by distinct thymic antigen-presenting cell subsets, assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expression levels were normalized to subsets previously reported to express the respective ligands: CCL17 and CCL22 expression were normalized to cDC2 , and CCL19 and CCL21 expression to mTEC lo ( ; ). Data were compiled from three independent experiments with triplicates. One-way ANOVA with Dunnett’s multiple comparisons correction was used for statistical analysis (mean ± SEM; ns: p > 0.05, **p < 0.01). ( F ) The percentage of WT or Ccr4 −/− T cells that proliferated at days 3 and 5 when cultured without splenocytes, with unstimulated splenocytes, or with lipopolysaccharide (LPS)-stimulated splenocytes, as indicated. Data were compiled from four independent experiments with triplicate wells, and unpaired t -tests with Holm–Šídák’s multiple comparisons correction was used for statistical analysis (mean ± SEM; *p < 0.05).

Article Snippet: peptide, recombinant protein , Recombinant Murine CCL21 , Peprotech , Cat # 250–13 , .

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture

Journal: eLife

Article Title: CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

doi: 10.7554/eLife.80443

Figure Lengend Snippet:

Article Snippet: peptide, recombinant protein , Recombinant Murine CCL21 , Peprotech , Cat # 250–13 , .

Techniques: Purification, Control, Flow Cytometry, Immunostaining, Plasmid Preparation, Sequencing, Recombinant, SYBR Green Assay, Software

Journal: Cell Death and Differentiation

Article Title: RAF kinases are stabilized and required for dendritic cell differentiation and function

doi: 10.1038/s41418-019-0416-4

Figure Lengend Snippet: Role of RAF kinases in moDC migration. a F-actin of LPS-stimulated moDCs, which were treated with LY3009120 (1 µM) or trametinib (1 µM), were stained with phalloidin and visualized by a Leica SP8 confocal microscope. b Morphology was analyzed by determining the circularity index of 26–60 cells per condition. c CCR7 mRNA levels were determined by real-time PCR after treating moDCs for 48 h with LY3009120 (1 µM) or trametinib (1 µM) in the presence of LPS ( n = 4). d The surface marker expression of CCR7 was analyzed by flow cytometry after treating moDCs with LY3009120 (1 µM) or trametinib (1 µM) while stimulating with LPS (100 ng/ml) and PGE 2 (1 µg/ml) ( n = 6). e Directed migration of moDCs towards the cytokine CCL21 (200 ng/ml) was determined in transwell migration experiments by counting successfully migrated cells after 3 h ( n = 5)

Article Snippet: The lower chamber was filled with serum-free media supplemented with 200 ng/ml recombinant CCL21 (human: Cat. No. 11343180, Immunotools; murine: Cat. No. 250–13, PeproTech) as a chemoattractant.

Techniques: Migration, Staining, Microscopy, Real-time Polymerase Chain Reaction, Marker, Expressing, Flow Cytometry

Journal: Cell Death and Differentiation

Article Title: RAF kinases are stabilized and required for dendritic cell differentiation and function

doi: 10.1038/s41418-019-0416-4

Figure Lengend Snippet: Role of RAFs on murine DC activation and migration. a BMDCs from C57BL/6 mice were treated with trametinib (1 μM) or with LY3009120 (1 μM) in presence or absence of LPS (100 ng/ml) for 48 h and surface marker expression (CD86, and CD80) was analyzed by flow cytometry. Shown are relative mean fluorescence intensities of multiple independent experiments. b Expression of the chemokine receptor CCR7 was investigated as described in a . c In vitro migration towards the cytokine CCL21 (200 ng/ml) was investigated with a transwell migration chamber. Successfully migrated BMDCs were determined after 3 h by MTT assay ( n = 4). d In vivo migration of labeled BMDCs towards auricular lymph nodes was monitored and quantified in mice as mentioned in the methods part ( n = 7). e The in vivo effect of LY3009120 was investigated on splenic DCs from C57BL/6J mice. The inhibitor (15 mg/kg; solved 0.5% CMC) was administered i.p. on day 0 and day 1. LPS (10 µg/mouse) was injected i.p. on day 2. Control mice received the corresponding vehicle. Six hours after LPS administration, spleens were isolated and surface expression of MHCII, CD86, and CD80 on DCs was analyzed by flow cytometry ( n = 4)

Article Snippet: The lower chamber was filled with serum-free media supplemented with 200 ng/ml recombinant CCL21 (human: Cat. No. 11343180, Immunotools; murine: Cat. No. 250–13, PeproTech) as a chemoattractant.

Techniques: Activation Assay, Migration, Marker, Expressing, Flow Cytometry, Fluorescence, In Vitro, MTT Assay, In Vivo, Labeling, Injection, Control, Isolation