Journal: iScience

Article Title: Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

doi: 10.1016/j.isci.2023.107172

Figure Lengend Snippet: Altered N -glycosylation profile of muscle biopsies at diagnosis associates with disease course and response to therapy in patients with IIM (A) Schematic representation of the N -glycosylation pathway from high-mannose N -glycans (recognized by GNA lectin) toward more complex N -glycans, with β1,6-branching antennae (detected by L-PHA lectin). (B) Representative images of skeletal muscle from IIM and HC, low L-PHA, and high GNA reactivity in IIM biopsies (scale bar = 100 μm). (C) Qualitative score of GNA and L-PHA staining intensity from total tissue from IIM and HC biopsies. Analysis performed for 22 IIM and 11 HC cases. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (D) Distribution of IIM disease cases in high (≥50%) and low (<50%) GNA reactivity, at each cellular component (muscular or stromal). Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (E) Quantitative analysis of staining intensity in each cellular compartment (muscular or stromal) of IIM compared with HC. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (F) MGAT1 and MAN2A1 mRNA expression levels on total muscle FFPE biopsies from patients with IIM and HC individuals. Each dot represents one biological sample. Relative quantification (RQ) of mRNA levels is expressed as mean ± SD (Mann-Whitney t-test: ∗p value<0.05, ∗∗p value<0.01). (G) ROC curve for the GNA reactivity from patients with IIM with poor disease course (more than 1 flare) and its respective AUC and p value. (H) The predictive capacity of high GNA reactivity and other clinic-pathological parameters to distinguish poor disease prognosis (that have more than 1 flare). (I) GNA reactivity ROC curve, respective AUC and p value, for IIM patients’ refractory to treatment. (J) The predictive capacity of high GNA reactivity and other clinic-pathological parameters to distinguish non-responder patients (that do not respond to therapy). The error bars represent the standard deviation of the data.

Article Snippet: L-PHA biotinylated , Vector Laboratories , Cat#B-1115.

Techniques: Staining, One-tailed Test, MANN-WHITNEY, Expressing, Standard Deviation

Journal: iScience

Article Title: Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

doi: 10.1016/j.isci.2023.107172

Figure Lengend Snippet: Altered N -glycosylation profile of CD4+ T cells associates with increased activation and cytokine production in patients with IIM (A) Cell surface β1,6-branching N -glycans (L-PHA) and high-mannose (GNA) N -glycans of CD4 + T cells from IIM and HC peripheral blood, analyzed by flow cytometry. Levels of median fluorescence intensity (MFI) from patients with IIM were normalized for the average of healthy control MFI. Fold change ratio is represented as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (B) MGAT1 and (C) MAN2A1 mRNA expression levels on peripheral CD3 + T cells from patients with IIM and HC individuals. Each dot represents one biological sample. Relative quantification (RQ) of mRNA levels is expressed as mean ± SD (Mann-Whitney t-test: ∗p value<0.05,∗∗p value<0.01). (D) Percentage of IL-6-producing CD4 + T cells in peripheral blood, gated on CD4 + T cells with high vs. low levels of L-PHA staining, from patients with IIM and HC individuals. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (E) Levels of total IL-6-producing CD4 + T cells, analyzed by flow cytometry. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (F) Levels of total IL-4-producing CD4 + T cells, analyzed by flow cytometry. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests. (G) Serum IL-6 quantification by ELISA from IIM and HC. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (H) Cell surface β1,6-branching N -glycans (L-PHA) of CD4 + T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (I) Percentage of activated (CD25 or CD69) CD4 + T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (J) Percentage of IL-6-producing CD4 + T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (K) Levels of IL-6 produced by CD3 − cells, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (L) Median fluorescence intensity (MFI) of DC-SIGN and mannose receptor (MR) in DCs and macrophages from IIM and HC. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). The error bars represent the standard deviation of the data.

Article Snippet: L-PHA biotinylated , Vector Laboratories , Cat#B-1115.

Techniques: Activation Assay, Flow Cytometry, Fluorescence, One-tailed Test, MANN-WHITNEY, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Produced, Standard Deviation

Journal: iScience

Article Title: Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

doi: 10.1016/j.isci.2023.107172

Figure Lengend Snippet: GlcNAc supplementation of fresh muscle biopsies from IIM patients attenuates pro-inflammatory immune response (A) Experimental outline of 150 mM N -acetylglucosamine (GlcNAc) supplementation of IIM biopsies for 72 h. Each patient biopsy was longitudinally cut into 2 similar portions, one for the supplementation (GlcNAc 150 mM) and other for control without supplementation (IIM). After incubation, cells were lysed for protein extraction and lectin blot analysis, together supernatant which was collected and released cytokines were analyzed by cytokine bead assay (CBA). (B) Adjusted values of intensity from L-PHA and GNA lectin blot of whole tissue cell lysate with or without GlcNAc supplementation. Normalized for loading control (actin). Levels are expressed as mean ± SD. Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (C) Fold change of cytokines secreted to the supernatant by IIM biopsies supplemented with 150 mM GlcNAc for 72 h, normalized by the respective basal condition. Levels of released cytokine were analyzed by cytokine bead assay (CBA) One-way ANOVA followed by Tukey’s post test.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Levels are expressed as mean ± SD.

Article Snippet: L-PHA biotinylated , Vector Laboratories , Cat#B-1115.

Techniques: Incubation, Protein Extraction, MANN-WHITNEY

Journal: iScience

Article Title: Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

doi: 10.1016/j.isci.2023.107172

Figure Lengend Snippet:

Article Snippet: L-PHA biotinylated , Vector Laboratories , Cat#B-1115.

Techniques: Plasmid Preparation, Recombinant, Random Hexamer Labeling, Protease Inhibitor, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: PLoS ONE

Article Title: β1,6 GlcNAc Branches-Modified PTPRT Attenuates Its Activity and Promotes Cell Migration by STAT3 Pathway

doi: 10.1371/journal.pone.0098052

Figure Lengend Snippet: (A) The level of GnT-V protein was detected in different cell lines by immunoblot. (B) The GnT-V-7721 or GnT-V-HT29 stable cells were identified by detecting the level of GnT-V protein using immunoblot. (C) GnT-V-7721 and Mock-7721 cells were seeded on coverslips, followed by fixation and staining with biotinylated L-PHA and FITC-conjugated avidin, and then visualized under fluorescence microscopy. (D) Flow cytometry was performed with biotinylated L-PHA in GnT-V-7721 and GnT-V-HT29 stable cells, as well as Mock cells. FITC-conjugated avidin D staining was used as negative control.

Article Snippet: Biotinylated phaseolus vulgaris leucoagglutinin (L-PHA), biotinylated datura stramonium lectin (DSL), agarose bound L-PHA, agarose bound streptavidin and fluorescein anti-avidin D were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Western Blot, Staining, Avidin-Biotin Assay, Fluorescence, Microscopy, Flow Cytometry, Negative Control

Journal: PLoS ONE

Article Title: β1,6 GlcNAc Branches-Modified PTPRT Attenuates Its Activity and Promotes Cell Migration by STAT3 Pathway

doi: 10.1371/journal.pone.0098052

Figure Lengend Snippet: (A) 4 types of RPTPs were evaluated in Mock-7721 and GnT-V-7721 using immunofluorescent staining. Cells were placed on coverslips overnight and stained with anti-PTPRM, anti-PTPRT, anti-PTPRK, or anti-PTPRU antibody, respectively. Then, FITC-labeled anti-mouse IgG or Cy3-labeled anti-rabbit IgG was used based on the specificity of the primary antibody. Intensity of fluorescent was visualized under confocal microscopy. (B) Lectin precipitation was performed with L-PHA bounded agarose, followed by immunoblot with anti-PTPRT antibody in GnT-V-7721 and Mock-7721. (C) PTPRT was immunoprecipitated in GnT-V-7721 and Mock-7721, and then subjected to the immunoblot with biotinylated L-PHA, biotinylated DSL and anti-PTPRT antibody, respectively.

Article Snippet: Biotinylated phaseolus vulgaris leucoagglutinin (L-PHA), biotinylated datura stramonium lectin (DSL), agarose bound L-PHA, agarose bound streptavidin and fluorescein anti-avidin D were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Staining, Labeling, Confocal Microscopy, Western Blot, Immunoprecipitation

Journal: Journal of Cancer

Article Title: Knockdown of C1GalT1 inhibits radioresistance of human esophageal cancer cells through modifying β1-integrin glycosylation

doi: 10.7150/jca.25252

Figure Lengend Snippet: X-ray irradiation induces the expression of C1GalT1 and core 1 O-glycans in esophageal cancer cells. (A) C1GalT1 mRNA expression in esophageal cancer cells 48 h after irradiation was measured by qPCR. (B) C1GalT1 protein expression in esophageal cancer cells 48 h after irradiation was measured by western blot. (C) Expression of core 1 O-glycans was detected by flow cytometry. (D) Core 1 O-glycans were analyzed by Jacalin lectin blot. (E) Expression of core 1 O-glycans in esophageal cancer cells 48 h after irradiation was measured by flow cytometry. *P<0.05.

Article Snippet: The blotted polyvinylidene (PVDF) membranes were incubated with antibodies against C1GalT1 (ab57492; Abcam, 1:1000), GAPDH (AF1186;Beyotime,1:2000), β1-integrin(ab24693; Abcam, 1:1000), Try397 pFAK (sc81493; Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:800), Ser 473 pAkt (sc293125; Santa Cruz Biotechnology, 1:800) and biotinylated Jacalin (B1115; Vector Laboratories, Burlingame, CA, 1:1000).

Techniques: Irradiation, Expressing, Western Blot, Flow Cytometry

Journal: Journal of Cancer

Article Title: Knockdown of C1GalT1 inhibits radioresistance of human esophageal cancer cells through modifying β1-integrin glycosylation

doi: 10.7150/jca.25252

Figure Lengend Snippet: C1GALT1 modifies O-glycans on β1-integrin. (A)Expression of core 1 O-glycans was detected by flow cytometry. (B) Core 1 O-glycans were analyzed by Jacalin lectin blot. (C) Cell lysates were pulled down (PD) by Jacalin and immunoblotted with an anti-β1-integrin antibody. (D) Cell lysates were immunoprecipitated with an anti-β1-integrin antibody and blotted with Jacalin or anti-β1-integrin antibody. **P<0.001.

Article Snippet: The blotted polyvinylidene (PVDF) membranes were incubated with antibodies against C1GalT1 (ab57492; Abcam, 1:1000), GAPDH (AF1186;Beyotime,1:2000), β1-integrin(ab24693; Abcam, 1:1000), Try397 pFAK (sc81493; Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:800), Ser 473 pAkt (sc293125; Santa Cruz Biotechnology, 1:800) and biotinylated Jacalin (B1115; Vector Laboratories, Burlingame, CA, 1:1000).

Techniques: Expressing, Flow Cytometry, Immunoprecipitation

Journal: Journal of Biomedicine and Biotechnology

Article Title: Explanting Is an Ex Vivo Model of Renal Epithelial-Mesenchymal Transition

doi: 10.1155/2011/212819

Figure Lengend Snippet: Lectin staining characteristics of cell outgrowths, 10 days after explanting. The majority of cells stain for Pha-E and Pha-L, lectins specific for the proximal tubular epithelium. (a) Arachis hypogaea (AH); (b) Bandeiraea simplicifolia I (BSL-I); (c) Phaseolus vulgaris erythroagglutinin (Pha-E); (d) Phaseolus vulgaris leukoagglutinin (Pha-L). Scale bar = 25 μ m.

Article Snippet: Tissue sections were incubated for 2 hr with biotin-conjugated phaseolus vulgaris leukoagglutinin (Pha-L; Vector) (proximal tubules and thick loop of Henle), phaseolus vulgaris erythroagglutinin (Pha-E; Vector) (proximal tubules), Bandeiraea simplicifolia I (BSL-I; Sigma) (collecting ducts, vasa recta ), or Arachis hypogaea (Sigma) (distal convoluted tubules and collecting ducts).

Techniques: Staining