Journal: Nature Communications

Article Title: Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis

doi: 10.1038/s41467-021-22365-z

Figure Lengend Snippet: a Illustration shows pathways involved in sialic acid and sialylated glycans biosynthesis. Relative expression for each gene was extracted from bulk RNA-Seq data comparing naive and arthritic SFs directly isolated from mouse synovium. b Synovial fibroblasts expanded from healthy or arthritic mouse joints were stimulated in vitro for 6 h with recombinant IL-1β, TNF, or IL-17A [10 ng/ml]. mRNA expression for genes involved in sialylation [as shown in a ] was evaluated by RT-qPCR. Il6 was included as a positive control to confirm cell activation. Volcano plots show log2 fold difference in stimulated cells ( x axis) and p -value ( y axis). Each dot represents the mean of three independent experiments analyzed in triplicate, with naive cells in blue and arthritic cells in red. The pink line indicates a twofold-change in gene expression threshold and blue lines indicate p = 0.05 threshold in the t -test to evaluate statistical significance. c TNF, but not IL-1β or IL-17A reduces α2-6-sialylation of synovial fibroblasts both at RNA ( St6gal1 expression) and protein (SNA binding) levels. Expression of St6gal1 (left panel ) was evaluated by RT-qPCR. Results show the mean of three independent experiments analyzed in triplicate, error bars represent SEM. Statistical significance was determined using two-tail unpaired t -tests where * p < 0.05 and ** p < 0.01. Actual p -values: a 0.0069, b 0.0277. Expression of α2-6 sialylated glycoconjugates was determined by SNA binding (right panel) as in Fig. . Results are merged from 3 (naive) and 2 (CIA) independent experiments, n = 10 (DMEM naive), 16 (IL-1 naive and IL-17 naive), 13 (TNF naive), 10 (DMEM CIA), 12 (IL-1 naive and IL-17 naive), and 9 (TNF CIA). Statistical significance was determined using two-tail unpaired t -tests, where ** p < 0.01 and *** p < 0.001. Actual p -values: c 0.0124, d 0.0091. d SFs were expanded from OA human synovium. Cells were stimulated with recombinant human TNF (10 ng/ml) for 6 h. IL6 , ST6GAL1, and ST6GAL2 expression were determined by qPCR. Results show relative expression to HPRT, showing mean ± SEM. Statistics: one-tail unpaired t -test, n = 3 biological replicates from cells pooled from three donors, *** p < 0.001, * p < 0.05. Actual p -values: e <0.0001, f 0.0170, g 0.0321. e Relative expression of individual sialylated glycan structures was evaluated by MALDI-TOF MS analysis as in Fig. . Ratios of sialylated vs non-sialylated twin structures were evaluated for non-stimulated cells (blue) and TNF stimulated (red) cells (48 h, 10 ng/ml). Results are from one single experiment using pooled cells from three animals.

Article Snippet: TaqMan predesigned probes (ThermoFisher Scientific) were used to evaluate mouse Actb (4352933E), Il6 (Mm00446190_m1 ), St6gal1 (Mn00486119_m1) , St6gal2( Mm01268915_m1) , St3gal1 (Mm00501493_m1) , St6galnac1 (Mm01252949_m1) , St6galnac3 (Mm01316813_m1) and Mmp3 (Mm00440295_m1) and human IL6( Hs00174131_m1) , ST6GAL1 (Hs00949382_m1) , ST6GAL2 (Hs00383641) and HPRT (4333768T).

Techniques: Expressing, RNA Sequencing, Isolation, In Vitro, Recombinant, Quantitative RT-PCR, Positive Control, Activation Assay, Gene Expression, Binding Assay, Glycoproteomics

Journal: Frontiers in Oncology

Article Title: Differential O - and Glycosphingolipid Glycosylation in Human Pancreatic Adenocarcinoma Cells With Opposite Morphology and Metastatic Behavior

doi: 10.3389/fonc.2020.00732

Figure Lengend Snippet: Immune recognition of glycan structures on PaTu-S and PaTu-T cells. (A) Interaction of immature DCs with PaTu-S and PaTu-T were visualized by fluorescence microscopy. Bar = 100 μm. (B) Binding of immature DCs to PaTu-S and PaTu-T in a cell adhesion assay, in the presence or absence of EGTA. Results are derived from 6 independent experiments using different donors and expressed as average percentage binding ± SEM. (C) Binding of recombinant human galectins Gal-1, Gal-3, and Gal-4 (5 μg/ml) to the PDAC cell lines was measured by flow cytometry. Results are given as average MFI ± SEM of at least 2 independent experiments. (D) Binding of Fc-chimeras of DC-SIGN, MGL, DCIR and Dectin-1 to PaTu-S and PaTu-T cells was measured by flow cytometry. Results are given as average MFI ± SEM of at least 3 independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Mouse anti-human galectin (Gal)-1 , and anti-Tn monoclonal antibodies were kindly provided by Dr. RD Cummings (Boston, USA), goat anti-human Gal-4 was purchased from R&D Systems (Minneapolis, MN) and anti-sialyl Lewis A/CA 19-9 monoclonal antibody was from LifeSpan Biosciences (Seattle, WA).

Techniques: Fluorescence, Microscopy, Binding Assay, Cell Adhesion Assay, Derivative Assay, Recombinant, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Enzyme Replacement Therapy Can Reverse Pathogenic Cascade in Pompe Disease

doi: 10.1016/j.omtm.2020.05.026

Figure Lengend Snippet: AT-GAA Reversed the Levels of the Autophagic Markers and Alleviated the Burden of Accumulated Protein Aggregates and ER-Stress in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group; n = 5 for western blot with LC3 antibody). (B) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with K63-linked ubiquitin specific antibody (n = 4 for each group). Western blot with anti-GAPDH and Ponceau S staining were used as loading controls. (C) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with anti-Grp 78 antibody (ER-stress marker; n = 4 for each group); GAPDH was used as loading control. Each data point represents an individual mouse. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: SQSTM1/p62 (#ab56416; mouse monoclonal), galectin 9 (#ab69630; rabbit polyclonal), Ubiquitin K48 (Linkage Specific) (#ab140601; rabbit monoclonal), and glyceraldehyde-3-phosphate dehydrogenase (#ab9485; rabbit polyclonal) were purchased from Abcam.

Techniques: Western Blot, Ubiquitin Proteomics, Staining, Marker, Control