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:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Interaction of Rex with either hCRM1 or rCRM1. (A) REF52 cells were transfected with the plasmid expressing either GAL-hCRM1 or GAL-rCRM1 in combination with pRexVP, pG5BLuc, and pCDMβ-gal. The Luc/β-Gal ratios of all samples were calculated. GAL-, plasmid expressing only the GAL4 region, which was used as a negative control. (B) Western blot of GAL-hCRM1 and GAL-rCRM1 proteins synthesized in transfected REF52 cells. (C) Pull-down assay using recombinant Rex protein. Purified His-Rex(g10) proteins immobilized on chelating Sepharose were incubated with HeLa or REF52 cell extract in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of GTP-charged recombinant RanQ69L protein. The sample of lane 1 contained neither cell lysate nor recombinant RanQ69L proteins during incubation. The volume of the cell extract subjected to the binding reaction was nine times of that of the input fraction.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Synthesized, Pull Down Assay, Recombinant, Purification, Incubation, Binding Assay

Journal:

Article Title: Reversible Sialylation: Synthesis of CMP-NeuAc from 5?-CMP using ?2,3-sialyl O-glycan, glycolipid and macromolecule based donors allow for the synthesis of diverse sialylated products

doi: 10.1021/bi701472g

Figure Lengend Snippet: * CMP-NeuAc but not UMP-NeuAc formed by reverse sialylation is an efficient sialyl donor for forward sialyltransferase reaction

Article Snippet: Materials Rat recombinant ST3Gal-II (α2,3(O)ST), ST3Gal-III (α2,3(N)ST) and ST6Gal-I (α2,6(N)ST) were purchased from Calbiochem ( 14 ).

Techniques: Activity Assay

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: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Immunohistochemistry staining of IL11RA and IL6R in healthy human liver sections (scale bars, 20 µm, n = 1 independent experiment, due to limited amount of human liver section). b Flow cytometry forward scatter (FSC) plots of IL11RA, IL6R, and gp130 staining and fluorescence intensity plots of IL11RA and IL6R staining on hepatocytes and THP-1. c Abundance of IL11RA1 and IL6R reads in hepatocytes at baseline based on RNA-seq (left) and Ribo-seq (right) (transcripts per million, TPM) ( n = 3). d , e Read coverage of d IL11RA1 and e IL6R transcripts based on RNA-seq (gray) and Ribo-seq (red) of primary human hepatocytes ( n = 3). f Western blots showing ERK, JNK, and STAT3 activation status and g ALT secretion ( n = 4) by hepatocytes following a dose range stimulation of either hyperIL11 or hyperIL6. h ALT levels in the supernatants of hepatocytes stimulated with hyperIL11 alone or in the presence of increasing amounts of soluble gp130 (sgp130) ( n = 4). i , j Western blots of hepatocyte lysates showing i phosphorylated ERK and JNK and their respective total expression in response to hyperIL11 stimulation alone or with sgp130 and j phospho-STAT3 and total STAT3 in response to hyperIL6 stimulation with and without sgp130. k Representative FSC plots of propidium Iodide (PI) staining of IL11-stimulated hepatocytes in the presence of sgp130 or soluble IL11RA (sIL11RA). l Western blots showing phospho-ERK, phospho-JNK, cleaved caspase-3, and their respective total expression, NOX4, and GAPDH in hepatocytes in response to IL11 stimulation alone or in the presence of sgp130 or sIL11RA. i , j , l Representative data of n = 2 independent experiments. b – l Primary human hepatocytes; f – l 24 h stimulation; hyperIL11, hyperIL6, IL11 (20 ng/ml), sgp130, sIL11RA (1 µg/ml). c , g , h Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). g , h One-way ANOVA with Dunnett’s correction. Source data are provided as a Source data file.

Article Snippet: Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human IL6 (206-IL-010, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems).

Techniques: Immunohistochemistry, Staining, Flow Cytometry, Fluorescence, RNA Sequencing, Western Blot, Activation Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a – l Data for palmitate (0.5 mM) loading experiment on primary human hepatocytes (24 h) in the presence of either IgG (2 µg/ml), anti-IL11RA (X209, 2 µg/ml), or sgp130 (1 µg/ml). a IL11, b IL6, c CCL2, and d CCL5 protein secretion levels as measured by ELISA of supernatants ( n = 3). e Representative FSC plots and f quantification of PI +ve hepatocytes stimulated with palmitate ( n = 3). g ALT levels in supernatants ( n = 3). h Total and reduced hepatocyte glutathione (GSH) levels ( n = 4). i Representative fluorescence images of DCFDA (2′,7′-dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100 µm) ( n = 4 independent experiments). j Western blots of phospho-ERK, ERK, phospho-JNK, JNK, cleaved caspase-3, caspase-3, NOX4, and GAPDH. Data from two independent biological experiments are shown. k Percentage of fatty acid oxidation by Seahorse assay ( n = 10). l Representative fluorescence images (scale bars, 100 µm) of ACTA2 +ve cells and Collagen I immunostaining for experiment shown in Supplementary Fig. ( n = 2 independent experiments, 14 measurements per condition per experiment). a – d , f , g Mean ± SD; h , k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). a – d , f – h , k One-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human IL6 (206-IL-010, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Staining, Western Blot, Immunostaining, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Schematic of HFMCD feeding regimen for AAV8-Alb-Cre injected Il11ra1 loxP/loxP (conditional knockout; CKO) mice for experiments shown in ( b – k ). Il11ra1 loxP/loxP mice were intravenously injected with either AAV8-Alb-Null or AAV8-Alb-Cre to delete Il11ra1 specifically in hepatocytes 3 weeks prior to the start of HFMCD diet. b Western blots of hepatic IL11RA and GAPDH ( n = 3 mice/group). c Body weight (shown as a percentage (%) of initial body weight). d Representative gross anatomy, H&E-stained (scale bars, 50 µm), and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 5 mice/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. e Hepatic triglycerides content. f Serum ALT levels. g Serum AST levels. h Hepatic GSH content. i Hepatic collagen levels. j Heatmap showing hepatic mRNA expression of pro-inflammatory markers ( Tnfα , Ccl2 , Ccl5 ) and fibrotic markers ( Col1a1, Col1a2, Col3a1, Acta2) . Values are shown in Supplementary Fig. and d. k Western blots showing hepatic ERK and JNK activation status ( n = 3 mice/group). c , e – j NCD ( n = 5 mice/group), HFMCD ( n = 6 mice/group). c Data are shown as mean ± SD, two-way ANOVA with Tukey’s correction, statistical significance ( P values) are shown for comparison between WT HFMCD and CKO HFMCD; e – i data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); two-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human IL6 (206-IL-010, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems).

Techniques: Injection, Knock-Out, Western Blot, Staining, Expressing, Activation Assay, Comparison, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Schematic of WDF-fed control and CKO mice for data shown in ( b – m ). Three weeks following AAV8-Alb-Null or AAV8-Alb-Cre virus injection, CKO mice were fed WDF for 16 weeks. b Western blots showing hepatic levels of IL11RA and GAPDH ( n = 3 mice/group). c Body weight (shown as a percentage (%) of initial body weight). d Fat mass. e Representative gross anatomy, H&E-stained (scale bars, 50 µm), and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 5/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. f Hepatic triglycerides content. g Liver weight. h Serum ALT levels. i Serum AST levels. j Hepatic GSH content. k Hepatic collagen levels. l Hepatic pro-inflammatory and fibrotic genes expression on heatmap (values are shown in Supplementary Fig. and d). m Western blots showing activation status of hepatic ERK and JNK ( n = 3 mice/group). c , d , f – l n = 5 mice/group. c , d Data are shown as mean ± SD, two-way ANOVA with Tukey’s correction, statistical significance ( P values) are shown for comparison between WT WDF and CKO WDF; f – k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); two-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human IL6 (206-IL-010, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems).

Techniques: Control, Virus, Injection, Western Blot, Staining, Expressing, Activation Assay, Comparison, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Schematic showing WDF feeding regimen of Il11ra1 +/+ (WT) and Il11ra1 −/− (KO) mice for experiments shown in ( b – n ). AAV8-Alb-Null, AAV8-Alb-mbIl11ra1 (full-length membrane-bound Il11ra1), and AAV8-Alb-sIl11ra1 (soluble form of Il11ra1)-injected KO mice were given 16 weeks of WDF feeding, three weeks following virus administration. b Western blots showing hepatic levels of IL11RA and GAPDH ( n = 2 mice/group). c Representative gross anatomy, H&E-stained (scale bars, 50 µm) and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 6 mice/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. d Liver weight. e Hepatic triglycerides content. f Serum ALT levels. g Serum AST levels. h Hepatic GSH content. i Hepatic collagen content. j Hepatic pro-inflammatory and fibrotic genes expression heatmap (values are shown in Supplementary Fig. and d). k Western blots showing activation status of hepatic ERK and JNK ( n = 2 mice/group). l Fasting blood glucose levels. m Serum triglycerides levels. n Serum cholesterol levels. d – j , l – n n = 6 mice/group. d – i , l – n Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); one-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human IL6 (206-IL-010, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems).

Techniques: Membrane, Injection, Virus, Western Blot, Staining, Expressing, Activation Assay, Whisker Assay

Journal: Nature Communications

Article Title: Secretory GFP reconstitution labeling of neighboring cells interrogates cell–cell interactions in metastatic niches

doi: 10.1038/s41467-023-43855-2

Figure Lengend Snippet: a t-Distributed stochastic neighbor embedding (t-SNE) plot of the GFP- hepatocytes from the healthy liver (control, n = 87) or the metastasized liver (distal, n = 59) and GFP+ hepatocytes from the metastasized liver (proximal, n = 99). Marker genes (two-sided version of Wilcoxon Rank-Sum test, adjusted p -value < 0.1, Log 2 FC > 0.25) among hepatocytes (proximal, distal, control) were used for clustering. Inserted numbers indicate cluster identification (left panel). The fraction rate of the hepatocytes in each cluster identified in the t-SNE plot (right panel). b Heatmap displaying expression level of clusters 2 and 3 marker genes of the top 20 ranked by Log 2 FC (two-sided version of Wilcoxon Rank-Sum test, adjusted p -value < 0.05) expressions in the GFP+ proximal hepatocytes. c The expression level of Lgals3 in the proximal, distal, and control. Data were statistically analyzed with analysis of variance and Tukey honestly significant difference test (95% family-wise confidence level). The adjusted p -values are shown in the graph. d Representative immunofluorescent staining images of galectin-3 in a metastasized liver section. Cancer cells (H2B-Azurite), hepatocytes (H2B-mCherry), and galectin-3 signals in the area of the hepatic tissue–metastasized colonies border (Liver–Meta border), distal liver tissue (Distal liver) and non-peripheral area of the metastatic colony (Non-peri meta) are shown. Similar results were observed in independent duplicate experiments. A scale bar indicates 20 µm. e Tumor spheroid proliferation assay of E0771 cells treated with galectin-3 in three-dimensional culture. Data were statistically analyzed with Holm-Sidak adjusted multiple t -test ( n = 102, 93, 90 and 72 for 0, 3, 5, 10 µg/mL galectin-3 respectively). The p -values are indicated in the graph. Data are presented as mean values ± SEM. Similar results were observed in independent duplicate experiments. Source data are provided as a Source Data file. f Transmigration assay of E0771 with recombinant galectin-3. Data were statistically analyzed with Holm-Sidak adjusted multiple t -test ( n = 3 biologically independent samples). The p -values are indicated in the graph. Data are presented as mean values ± SEM. Similar results were observed in independent duplicate experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining, the fixed sections were washed with PBS containing 0.05% Tween-20 (Nacalai Tesque) (PBS-T) three times and then were incubated in blocking buffer (5% donkey serum, FUJIFILM Wako Pure Chemical Corporation) for 1 h at room temperature, and then incubated with anti-mouse galectin-3 primary antibody (CL8942AP, 1:2000, Cedarlane Laboratories, Burlington, Canada) diluted in PBS containing 1% bovine serum albumin (Nacalai Tesque) overnight at 4 °C.

Techniques: Marker, Expressing, Staining, Proliferation Assay, Transmigration Assay, Recombinant