Journal: mBio

Article Title: Burkholderia pseudomallei Penetrates the Brain via Destruction of the Olfactory and Trigeminal Nerves: Implications for the Pathogenesis of Neurological Melioidosis

doi: 10.1128/mBio.00025-14

Figure Lengend Snippet: B. pseudomallei penetrates degraded olfactory and intact respiratory epithelium (RE). All sections were immunolabeled with anti- B . pseudomallei antibodies (green) and DAPI (blue); some were also labeled with anti-OMP antibodies and UEA1 lectin as indicated. (A) A coronal section of the nasal cavity (NC) shows that one side has extensive infection (arrow) while the other side has little evidence of infection. Boxed areas are shown in panels F to H as indicated. (B) A higher-magnification view of uninfected olfactory epithelium (OE) shows a uniform structure. (C) OE in an inoculated mouse shows an extensive presence of B. pseudomallei (arrow; green) in the NC at 24 h. The OE is crenellated (arrow with tail); the thin respiratory epithelium (RE) in the ventral NC was not visually affected. The asterisk indicates nonspecific autofluorescence. (D) Bacteria (green) occasionally penetrated relatively intact epithelium, but only in patches where neurons (immunolabeled with OMP; red) were absent (arrow). (E) In the olfactory epithelium, bacteria (green; arrow) were not associated with Bowman’s glands (labeled with UEA1 lectin; white; arrow with tail); olfactory neurons (red) are labeled with anti-OMP antibodies. (F to H) Higher-magnification views of the boxed areas indicated in panel A. (F) B. pseudomallei (arrow) was present on the surface of the OE, but no morphological reaction was apparent. (G) Ulceration of the OE (dashed line) was seen, although the presence of bacteria was limited (arrow). (H) The OE showed extensive destruction and loss of integrity, and bacteria were present (arrows) within the epithelium. Bacteria were not detected in the lamina propria (LP) underlying the OE (G and H). (I) In patches of respiratory epithelium, there was widespread infection with B. pseudomallei (arrow), but bacteria did not penetrate the deeper layers. (J and K) OMP immunolabeling (red) demonstrates that healthy epithelium was not penetrated by bacteria (arrow) despite their presence in the adjacent nasal cavity, but that epithelium was penetrated as the neurons partially degraded; panel K shows the same section as that in panel J but with the red channel (OMP) turned off. (L to O) OMP immunolabeling became patchy with some areas showing low levels of OMP reactivity (arrow with tail). Bacteria penetrated the outer layers and were present in nerve bundles in the lamina propria (arrows in panels N and O); panels M and O show the same sections as those in panels L and N, respectively, but with the red channel (OMP) turned off. (P and Q) Complete loss of the neuronal layer led to colonization of the remaining layer by bacteria (arrows); arrows with tails point to neurons in the nasal cavity and remaining epithelium. (R to T) Schematics summarizing the infection of the epithelium. (R) Sagittal view of the nasal cavity, olfactory bulbs (OB), and cortex (Cx). (S) In uninfected mice, the olfactory epithelium is uniform and neurons (red) are distributed throughout the epithelium. (T) When B. pseudomallei (green) is present, the majority of epithelium becomes crenellated but neurons remain within the epithelium and bacteria cannot penetrate. In some regions, the neurons are lost (arrow) and bacteria penetrate the remaining layers. Bar sizes are in μm.

Article Snippet: The biotinylated lectin UEA1 (1:100; Dako) was incubated similarly to the antibodies, followed by streptavidin-Alexa Fluor 647 (1:400; Invitrogen).

Techniques: Immunolabeling, Labeling, Infection, Bacteria

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

doi: 10.4049/jimmunol.1203086

Figure Lengend Snippet: a) Thymic architecture in WT129 and STAT1-/- mice as revealed by H&E staining of thymic sections (left panels); Keratin 5 (red) and UEA-1 (green) staining (middle panels), bar = 100μm; EpCAM (red) and AIRE (green) staining (right panels), bar = 50μm; M, medulla; C, cortex. b) Low magnification (4x) of H&E stains. c) Ratio of medullary to cortical cellularity in thymic sections from WT and STAT1-/- mice (data collected using ImageJ (NIH) and represent avg +/-SD of 3 sections each from 3 mice (p<0.05). d) AIRE+ cells per unit area (quantified from thymus sections shown in Fig.1a, lower panels) e) mTEC populations in WT and STAT1-/- thymi as determined by flow cytometric expression analysis of MHCII and UEA-1 (left), as well as AIRE and EpCAM (middle) on CD45lo stromal cells; MHCII and CD80 expression on UEA-1hi gated stromal cells. f) mTEC populations in WT and STAT1-/- thymi (n=4) using the indicated markers.

Article Snippet: Immunohistochemistry Paraformaldehyde-fixed 6 μm frozen sections were blocked with 1% FBS in PBS pH 7.4 for 20 min before staining with the indicated antibodies for 2 h or overnight: biotin labeled UEA-1 (Vector labs, Burlingame, CA), anti-AIRE (Santa Cruz, Santa Cruz, CA), Keratin 5 (Covance, Emeryville, CA), and Ep-CAM (G8.8, BD Biosciences).

Techniques: Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

doi: 10.4049/jimmunol.1203086

Figure Lengend Snippet: a) AIRE, INS2, and CRP mRNA levels in thymic stromal cells from WT and STAT1-/- mice (avg+/-sd; n=4) b) Thymic epithelial cells from WT mice (left) or TE-71 cells (right) were stimulated with 500 ng/mL RANKL for the indicated times and IFNβ mRNA levels were determined by qPCR (n = 4). c) Thymic epithelial cells were left untreated or pretreated with 1000 U/mL IFNβ prior to stimulation with 500 ng/mL RANKL for 24 hrs. UEA-1+ cells were analyzed for intracellular AIRE expression by flow cytometry. d) Thymic epithelial cells were depleted of CD80+/hi cells and then stimulated with 500 ng/mL RANKL, 1000 U/mL IFNβ or both for 24hrs. Lysates were probed for AIRE, ISG15, IRF7, cFOS, p65, and Traf6 by Western blotting (representative of three experiments). e) TE-71 cells were treated as indicated, and mRNA levels for AIRE and ISG15 were determined by qPCR (avg+/-sd; n=3). f) Thymic epithelial cells were treated as indicated and mRNA levels for CRP and INS2 were determined by qPCR (avg+/-sd; n=4).

Article Snippet: Immunohistochemistry Paraformaldehyde-fixed 6 μm frozen sections were blocked with 1% FBS in PBS pH 7.4 for 20 min before staining with the indicated antibodies for 2 h or overnight: biotin labeled UEA-1 (Vector labs, Burlingame, CA), anti-AIRE (Santa Cruz, Santa Cruz, CA), Keratin 5 (Covance, Emeryville, CA), and Ep-CAM (G8.8, BD Biosciences).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

doi: 10.4049/jimmunol.1203086

Figure Lengend Snippet: a) Flow cytometric analysis of YFP expression in total cells from thymus of WT (top left panel) or IFNβMOB/MOB mice (top right panel). Lower panels show UEA-1 and MHCII expression in YFP- (left panel) and YFP+ (right panel) cells in thymi of IFNβMOB/MOB mice. b) Stromal cells were purified from thymi of IFNβMOB/MOB mice and treated with anti-CD40 (1:100 dilution of supernatant), anti-LTβR (500ng/ml) or RANK ligand (500ng/ml) for 24 hours. Histogram illustrates IFNβ:YFP expression in UEA-1+ cells following the indicated treatments. Data is representative of three separate experiments.

Article Snippet: Immunohistochemistry Paraformaldehyde-fixed 6 μm frozen sections were blocked with 1% FBS in PBS pH 7.4 for 20 min before staining with the indicated antibodies for 2 h or overnight: biotin labeled UEA-1 (Vector labs, Burlingame, CA), anti-AIRE (Santa Cruz, Santa Cruz, CA), Keratin 5 (Covance, Emeryville, CA), and Ep-CAM (G8.8, BD Biosciences).

Techniques: Expressing, Purification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

doi: 10.4049/jimmunol.1203086

Figure Lengend Snippet: a) Schematic diagram of the interferon response reporter mice. Interferon-dependent Cre expression results in the excision of the mT sequence coding RFP and expression of mG encoding GFP (Muzumdar, et al. (27)). b) RFP and GFP expression in sections of thymi from WT and IFNAR1-/- mice carrying mT/mG and Mx1-Cre transgenes (upper panels, bar = 500μm). Sections were also stained for UEA-1 (lower panels, pseudo-colored purple, bar = 100μm). c) Flow cytometric analysis of CD45loUEA-1+ mTEC cells (upper panels) and CD45hiUEA-1- hematopoietic cells (lower panels) from thymi of either Mx1-Cre- or Mx1-Cre+ mT/mG mice. d) Histogram comparing GFP expression levels in CD45loUEA-1+ mTECs of Mx1-Cre- or Mx1-Cre+ mT/mG mice; arrow indicates GFP levels in CD45hi hematopoietic cells.

Article Snippet: Immunohistochemistry Paraformaldehyde-fixed 6 μm frozen sections were blocked with 1% FBS in PBS pH 7.4 for 20 min before staining with the indicated antibodies for 2 h or overnight: biotin labeled UEA-1 (Vector labs, Burlingame, CA), anti-AIRE (Santa Cruz, Santa Cruz, CA), Keratin 5 (Covance, Emeryville, CA), and Ep-CAM (G8.8, BD Biosciences).

Techniques: Expressing, Sequencing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

doi: 10.4049/jimmunol.1203086

Figure Lengend Snippet: a) Thymic stromal cells from WT or IRF7-/- mice were purified and stimulated with 500 ng/mL RANKL for the indicated time points. Expression of IFNβ and IκBα mRNAs was determined by qPCR. b) Thymic architecture in WT and IRF7-/- mice revealed by H&E staining of thymic sections (bar = 500 μm) c) Ratio of medullary to cortical cellularity in thymic sections from WT and IRF7-/- mice (data collected using ImageJ (NIH) and represent avg +/-sd of 3 sections each from 3 mice (p<0.05). d) mRNA levels of IFNβ, Iκbα, and ISG54 in thymic stromal cells from WT or IRF7-/- mice as determined by qPCR. e) Thymic architecture in WT and IRF7-/- mice as revealed by H&E staining of thymic sections (left panels), Keratin 5 and UEA-1 expression (middle panels, bar = 100 μm), UEA-1 only, as well as EpCAM and AIRE expression (right panels, bar = 50 μm) f) Relative mRNA levels of AIRE and INS2 in purified stromal cells from WT and IRF7-/-thymi measured by qPCR (n = 3). g) Histogram shows percentage of AIRE+ cells in the CD45loEpcam+UEA-1hi gate of thymic stromal cells from WT and IRF7-/- mice. h) Total number of UEA-1hi cells as determined by flow cytometric analysis of CD45loEpCAM+UEA-1hi mTEC populations from WT and IRF7-/- thymi. i) Total number of AIRE+ cells based on flow cytometric analysis of CD45loEpCAM+UEA-1hiAIRE+ mTEC populations in WT and IRF7-/- thymi (n = 4). j) Hypothetical model of IFNβ production and function in the thymus.

Article Snippet: Immunohistochemistry Paraformaldehyde-fixed 6 μm frozen sections were blocked with 1% FBS in PBS pH 7.4 for 20 min before staining with the indicated antibodies for 2 h or overnight: biotin labeled UEA-1 (Vector labs, Burlingame, CA), anti-AIRE (Santa Cruz, Santa Cruz, CA), Keratin 5 (Covance, Emeryville, CA), and Ep-CAM (G8.8, BD Biosciences).

Techniques: Purification, Expressing, Staining

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Hypofucosylation of Unc5b regulated by Fut8 facilitates macrophage migration. A , C The interaction between Unc5b and AAL, LCA, PHAL, VVL, MAL1, SNA detected by IP assays after ox-LDL treatment for 24 h. B , D Quantification of Unc5b expression from IP assay. Data are reported as the mean ± S.D. (n＞3, t-test). E HEK293T cells were pretreated with pCMV-Unc5b-GFP plasmid or pCMV-Unc5bko222,347-GFP plasmid and pCMV-Fut8-mCherry plasmid and cotransfected with pCMV-Fut8-mCherry plasmid, followed by immunostaining. ER (blue), Fut8 (red), Unc5b (green), merge (yellow)(bar=20 μm, n=4). F The α-1,6-fucosylated protein level of Unc5b was detected by LCA in cells with Fut8eo or Fut8siRNA plasmid treatment and the quantification of protein expression was shown below the blot. Data are reported as the mean ± S.D. (n＞3, *, p< 0.05vs NC group, t-test).

Article Snippet: Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK).

Techniques: Migration, Expressing, Plasmid Preparation, Immunostaining

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Fut8 participates in macrophage migration induced by ox-LDL. A The fucosylation level of the total protein of Raw 264.7 cells was determined by lectin blot after treatment with (0-75) mg/ml ox-LDL for 24 h. B Lectin blotting assays conducted to characterize the levels of total protein α-2,6 sialylation (SNA) and α-2,6 sialylation (MALI) levels in Raw 264.7 cells subjected to (0-75) mg/ml ox-LDL for 24 h. C The mRNA levels of Fut4, Fut7 and Fut8 in Raw 264.7 cells were analyzed by real-time RT-PCR after treatment with 0-75 mg/ml ox-LDL for 24 h. D The levels of total protein α-1,2-fucosylated (UEA1), α-1,3/4-fucosylated (LTL) and α-1,6-fucosylated (LCA) of Raw 264.7 cells were determined by lectin blot after treatment with 0-75 mg/ml ox-LDL for 24 h; E Western blotting was used to detect the expression of Fut8 protein in Raw 264.7 cells and mouse peritoneal macrophages after treatment with ox-LDL for 24 h. F , G Raw 264.7 cells were pretreated with siFut8 or pcDNA3.1-Fut8 plasmid, and then the number of cells on the dark side was identified by Transwell assay upon ox-LDL (50 μg/mL) treatment (bar=100 μm), the wound healing assay was used to identify the cell migration speed (bar=500 μm) or (H) the cells were stained for F-actin using Phalloidin-iFluor 488, and nuclei were counterstained with DAPI. Green:F-actin staining; Blue: DNA staining (bar=20 μm). Data were reported as the mean ± S.D. ( n=4, * p< 0.05 ; *** p< 0.001, t-test.).

Article Snippet: Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Transwell Assay, Wound Healing Assay, Staining

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Hypofucosylation of Unc5b regulated by Fut8 facilitates macrophage migration. A , C The interaction between Unc5b and AAL, LCA, PHAL, VVL, MAL1, SNA detected by IP assays after ox-LDL treatment for 24 h. B , D Quantification of Unc5b expression from IP assay. Data are reported as the mean ± S.D. (n＞3, t-test). E HEK293T cells were pretreated with pCMV-Unc5b-GFP plasmid or pCMV-Unc5bko222,347-GFP plasmid and pCMV-Fut8-mCherry plasmid and cotransfected with pCMV-Fut8-mCherry plasmid, followed by immunostaining. ER (blue), Fut8 (red), Unc5b (green), merge (yellow)(bar=20 μm, n=4). F The α-1,6-fucosylated protein level of Unc5b was detected by LCA in cells with Fut8eo or Fut8siRNA plasmid treatment and the quantification of protein expression was shown below the blot. Data are reported as the mean ± S.D. (n＞3, *, p< 0.05vs NC group, t-test).

Article Snippet: Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK).

Techniques: Migration, Expressing, Plasmid Preparation, Immunostaining

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Fut8 participates in macrophage migration induced by ox-LDL. A The fucosylation level of the total protein of Raw 264.7 cells was determined by lectin blot after treatment with (0-75) mg/ml ox-LDL for 24 h. B Lectin blotting assays conducted to characterize the levels of total protein α-2,6 sialylation (SNA) and α-2,6 sialylation (MALI) levels in Raw 264.7 cells subjected to (0-75) mg/ml ox-LDL for 24 h. C The mRNA levels of Fut4, Fut7 and Fut8 in Raw 264.7 cells were analyzed by real-time RT-PCR after treatment with 0-75 mg/ml ox-LDL for 24 h. D The levels of total protein α-1,2-fucosylated (UEA1), α-1,3/4-fucosylated (LTL) and α-1,6-fucosylated (LCA) of Raw 264.7 cells were determined by lectin blot after treatment with 0-75 mg/ml ox-LDL for 24 h; E Western blotting was used to detect the expression of Fut8 protein in Raw 264.7 cells and mouse peritoneal macrophages after treatment with ox-LDL for 24 h. F , G Raw 264.7 cells were pretreated with siFut8 or pcDNA3.1-Fut8 plasmid, and then the number of cells on the dark side was identified by Transwell assay upon ox-LDL (50 μg/mL) treatment (bar=100 μm), the wound healing assay was used to identify the cell migration speed (bar=500 μm) or (H) the cells were stained for F-actin using Phalloidin-iFluor 488, and nuclei were counterstained with DAPI. Green:F-actin staining; Blue: DNA staining (bar=20 μm). Data were reported as the mean ± S.D. ( n=4, * p< 0.05 ; *** p< 0.001, t-test.).

Article Snippet: Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Transwell Assay, Wound Healing Assay, Staining