uea1 fitc conjugated lectin  (Vector Laboratories)

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a – g , Representative immunofluorescence images of palatine tonsil sections from adult patients with OSA stained with the indicated antibodies and analyzed by confocal microscopy. a , Transition zone from the surface to the crypt epithelium. Stratified squamous epithelial cells at the surface (arrows) and UEA1 + epithelial cells infiltrated with CD3 + lymphocytes (arrowheads) at the transition area to the crypt epithelium are highlighted. DAPI, 4,6-diamidino-2-phenylindole. b , High-resolution analysis of the non-keratinized stratified squamous surface epithelium. PDPN-expressing basal cuboidal epithelial cells are highlighted (arrowheads). c , Morphology of the lymphoreticular crypt epithelium. Broadened PDPN-expressing basal layer of the crypt epithelium (arrows) and papillary extension providing access for blood vessels (asterisk) are highlighted. d , Epithelial and subepithelial (arrowheads) areas underpinned by PDPN + stromal cells. Boxed area shows the magnified epithelial–lymphoid tissue interface. e , B cell follicles with GCs and mantle zones (MZ) underpinned by PDPN + FRCs. f , CD3 + lymphocytes in epithelial, GC (asterisk) and interfollicular T cell (arrowhead) areas. g , T cell area surrounding B cell follicles (Fo) underpinned by PDPN + FRCs (arrowhead) and ACTA2 + PDPN − VSMCs (arrow). Square boxes show cell networks at higher magnification, and rectangular boxes indicate the regions of PDPN- and ACTA2-intensity measurements shown in h . h , Average pixel intensity of ACTA2 and PDPN signal-intensity measurements. The x axis represents the horizontal distance through the selection (rectangular boxes in g ), and the y axis represents the vertically averaged pixel intensity. The arrow highlights ACTA2 + PDPN + myofibroblasts in the perivascular space. Microscopy images are representative for n = 3 adult patients with OSA.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Selection, Microscopy

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a - f , Representative immunofluorescence images of palatine tonsil sections from adult patients with OSA stained with the indicated antibodies and analyzed by confocal microscopy. a , Structure of the palatine tonsil. UEA1 staining of the lymphoreticular epithelium highlights deep crypts permeating palatine tonsils. b , d , High resolution analysis of the non-keratinized stratified squamous surface epithelium. PDPN-expressing basal cuboidal epithelial cells are highlighted (arrowheads). c , e , Morphology of the lymphoreticular crypt epithelium. Broadened PDPN-expressing basal layer of the crypt epithelium is highlighted (arrows). f , Lymphoreticular crypt epithelium occupied by CD11c + myeloid cells. Boxed area shows magnified intraepithelial CD11c + cells surrounding a blood vessel (asterisk). Microscopy images are representative for n = 3 adult patients with OSA.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Microscopy

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , Representative UMAPs of distinct tonsillar stromal cell types according to the PhenoGraph clustering algorithm based on forward scatter (FSC)-A, side scatter (SSC)-A, CD31, ACTA2, PDPN and UEA1 flow cytometry data of CD45 − CD235a − cells ( n = 3 pediatric and n = 3 adult patients with OSA). Epithelial cells (EpCs), ACTA2 + cells, BECs, LECs, FRCs and negative cells (N) are indicated. b , UMAPs show the expression pattern of the indicated markers. c – e , Quantification of the indicated stromal cell types as a percentage of CD45 − CD235a − live cells in pediatric ( n = 5) and adult ( n = 6) patients with OSA. Data show average values of left and right tonsils for each patient. Mean and s.d. are indicated. P values were calculated with the two-sided Mann–Whitney test.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , b , PI16-positive cells in the subepithelial niche of tonsils from adult patients with OSA or tonsillitis. Cryosections were stained with the indicated antibodies and analyzed by confocal microscopy. c , High-resolution analysis of PI16 + cells shows intracellular PI16 signal in FBLN1 + reticular cells. Sections were stained with anti-FBLN1 and anti-PI16, and data were acquired by confocal microscopy and reconstructed in 3D. d , Morphology of the FBLN1 + subepithelial compartment in adult tonsils. Histological sections were stained with the indicated antibodies, and data were acquired and analyzed by confocal microscopy. The boxed area shows an FBLN1 + reticular network around UEA1 + endothelial cells at higher magnification on the right. e , High-resolution reconstruction of the adult subepithelial FBLN1 + FRC niche occupied by CD3 + and CD20 + lymphocytes. Sections were stained with the indicated antibodies, and data were acquired by confocal microscopy and reconstructed in 3D. f , Surface contact areas (blue) of FBLN1 + cells with CD20 + (arrows) and CD3 + (arrowheads) cells are shown at higher magnification. Microscopy images are representative for n = 3 adult patients with OSA and n = 3 adult patients with tonsillitis.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Staining, Confocal Microscopy, Microscopy

Journal: Science signaling

Article Title: The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

doi: 10.1126/scisignal.aac6479

Figure Lengend Snippet: (A) qRT-PCR analysis for FUK or PKCε expression in WM793 cells transfected with (left) EV, ATF2-targeted shRNA (shATF2), or shATF2 and ATF2WT, ATF2T52A, orATF2T52E;or(right)control (siCTL) or PKCε-targeted siRNA (siPKCε). (B) qRT-PCR for FUK mRNA expression (left) and immunoblotting for FUK protein abundance (right) in primary human melanocytes (HEMn), VGP melanoma cell lines (WM793, WM1346, and WM1366), metastatic melanoma cell lines (LU1205, 501Mel, and YUGASP), and transformed HEK293 cells. pATF2, phosphorylated ATF2; tATF2, total ATF2. (C) Top: FUK 5′ promoter with ATF2 binding sites (E1, E2, and E3), ChIP targets, and transcriptional start site. Bottom: ATF2 ChIP of the FUK 5′ promoter E1 and E2/3 targets in 501Mel (left) and LU1205 (right) cells. IgG, immunoglobulin G. (D) Wild-type (WT) or E1, E2, or E3 single-mutant FUK promoter luciferase activities in (left) WM793 cells transfected with EV (black) or ATF2T52E (gray) or in (right) 501Mel cells. (E) Fluorescence-activated cell sorting (FACS) analysis of UEA1 lectin binding of cell lines in (B).Graphic inset represents a glycan recognized by UEA1 (red triangles: α-1,2/1,3-fucose; dark gray squares: N-acetylglucosamine; light gray circles: galactose). (F) UEA1 FACS analysis in WM793 cells transfected with (left) EV, ATF2T52E, or caPKCε or (right) control or FUK-targeted shRNA (shFUK). (G) FACS analysis of LCA and PSA in WM793 cells transfected as indicated. Graphic inset represents a glycan recognized by LCA or PSA (red triangles: α-1,4/1,6-fucose). (H) A schematic of the fucose salvage and de novo synthesis pathways. All data are means ± SD from three experiments. *P < 0.05, **P < 0.005, ***P < 0.0005 by a standard t test compared with controls.

Article Snippet: The FITC-conjugated lectins UEA1, LCA, and PSA were purchased from Vector Laboratories.

Techniques: Quantitative RT-PCR, Expressing, Transfection, shRNA, Western Blot, Transformation Assay, Binding Assay, Mutagenesis, Luciferase, Fluorescence, FACS

Journal: Science signaling

Article Title: The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

doi: 10.1126/scisignal.aac6479

Figure Lengend Snippet: (A) qRT-PCR analysis of PKCε and mFuk expression in SW1 and K1735 cells. (B and C) Scratch assays assessing the migration of SW1 cells (B) in which cultures were supplemented with control water (Control; 5 μl) or L-fucose (L-Fuc; 50 μM) for 36 hours or (C) in which cells were transfected with EV or mFuk. (D) Two representative spheroids (n = 3 experiments) formed by EV- or FUK-expressing SW1 cells in culture. Scale bars, 10 μm. (E and F) Tumor volume curves (left) and lung metastasis counts (right) of (E) C3H/HeJ mice bearing implanted SW1 tumors or (F) mice bearing implanted EV-or mFuk-expressing SW1 tumors provided with control water (Control) or water supplemented with L-fucose (Fucose,100mM).Data are means ± SD from six mice per group. (G) Representative UEA1-low (upper) and UEA1-high (lower) histospots from a human melanoma TMA (n = 320) immunostained with UEA1 lectin (green), S100/HMB45 (red), and 4′,6-diamidino-2-phenylindole (DAPI, blue). (H) Distribution of UEA1 scores within S100/HMB45-positive melanoma cells of individual histospots from primary or metastatic melanoma samples. Means, SD, and n as indicated; P = 0.0002 by unpaired t test. Diamonds depict means ± 1 SD. (I) Kaplan-Meier survival curves according to UEA1 abundance in a melanoma patient cohort. Data in (A) to (C) are means ± SD from three experiments. *P < 0.05, **P < 0.005 by a standard t test compared with parental K1735 cell lines, controls, or starting time point.

Article Snippet: The FITC-conjugated lectins UEA1, LCA, and PSA were purchased from Vector Laboratories.

Techniques: Quantitative RT-PCR, Expressing, Migration, Transfection