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: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: Glucocorticoid‐mediated suppression of hypoxia‐induced galectin‐1/ LGALS1 expression in human Müller glial cells. (A) Müller glial cells were pretreated with aldosterone (Ald, 1 μmol/L), dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h, and LGALS1 gene expression levels were analysed. (B‐D) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h, and galectin‐1 protein expression levels in culture medium (B) and cell lysate (C, D) were analysed by ELISA (B, C) and immunoblot analysis (D). (E) Müller glial cells were pretreated with the glucocorticoid receptor antagonist RU486 (1 μmol/L) for 30 min before culture with Dex (1 μmol/L) and TA (1 μmol/L) in hypoxia (1% O 2 ) for 24 h, and LGALS1 gene expression levels were analysed. * P < .05, ** P < .01, n = 6 per group

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: Glucocorticoid‐mediated reduction of HIF‐1α protein and DNA‐binding activity with no impact on LGALS1 mRNA stability. (A) After Müller glial cells were cultured in hypoxia (1% O 2 ) for 24 h, the transcription inhibitor actinomycin D (2.5 μg/mL) with or without dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) was added and cells were harvested at the indicated times. RNA was extracted for real‐time qPCR analysis of LGALS1 . (B) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h, and HIF‐1α protein expression levels were analysed. (C, D) Müller glial cells were transfected with the control reporter pRL‐CMV, together with consensus HRE‐luciferase reporter (C) or human LGALS1 promoter‐luciferase reporter (D). Transfected Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h and assayed for luciferase activities. (E) Müller glial cells were cultured in hypoxia (1% O 2 ) for 1 h before harvest of samples. Binding of HIF‐1α to HREs in the LGALS1 promoter region was analysed by ChIP‐qPCR. (F) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h, and HIF1A gene expression levels were analysed. * P < .05, ** P < .01, n = 4‐6 per group

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques: Binding Assay, Activity Assay, Cell Culture, Expressing, Transfection, Luciferase

Journal: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: Glucocorticoid‐transactivated TSC22D3 interaction with and ubiquitination of HIF‐1α leading to suppression of hypoxia‐induced galectin‐1/ LGALS1 expression. (A) Müller glial cells were pretreated with the glucocorticoid receptor antagonist (RU486, 1 μmol/L) for 30 min before culture with dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) in hypoxia (1% O 2 ) for 24 h, and TSC22D3 gene expression levels were analysed. (B) After hypoxic stimulation (1% O 2 ) for 24 h, co‐IP of human Müller glial cell extracts using anti‐TSC22D3 and anti‐HIF‐1α antibodies was performed, followed by immunoblot analyses for HIF‐1α and TSC22D3. (C) Müller glial cells were incubated in hypoxia (1% O 2 ) with or without TA in the presence of the proteasome inhibitor MG132 (10 μmol/L) for 24 h. After co‐IP of cell extracts with anti‐HIF‐1α antibody, ubiquitinated HIF‐1α was detected using anti‐ubiquitin (Ub) antibody in TA‐treated cell extracts. (D, E) TSC22D3 (D) and LGALS1 (E) mRNA expression levels in human Müller glial cells exposed to control‐ or TSC22D3 ‐siRNA combined with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h. (F) Galectin‐1, HIF‐1α and TSC22D3 protein expression levels in human Müller glial cells exposed to control‐ or TSC22D3 ‐siRNA combined with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O 2 ) for 24 h. (G) Müller glial cells transfected with control‐ or TSC22D3 ‐siRNA were incubated in hypoxia (1% O 2 ) with or without TA in the presence of MG132 (10 μmol/L) for 24 h. After co‐IP of cell extracts with anti‐HIF‐1α antibody, ubiquitinated HIF‐1α was detected using anti‐Ub antibody. * P < .05, ** P < .01, n = 6 per group

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Incubation, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: Glucocorticoid‐mediated inhibition of diabetes‐induced retinal galectin‐1 and HIF‐1α together with transactivation of TSC22D3 in mice. (A‐C) Retinal Lgals1 (A) and Tsc22d3 (B) expression in mice with streptozotocin (STZ)‐induced diabetes at 2 mo. Dexamethasone (Dex, 50 pmol/eye) or triamcinolone acetonide (TA, 50 pmol/eye) were injected intravitreally to STZ mice, followed by mRNA (A, B) and protein (C) expression analyses after 24 h. (C) Immunoblot analyses for galectin‐1, TSC22D3 and HIF‐1α in the retina of diabetic mice treated with Dex or TA. * P < .05, ** P < .01, n = 6‐8 per group

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques: Inhibition, Expressing, Injection, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: Tissue co‐localization of HIF‐1α and galectin‐1 in glial cells in the epiretinal fibrovascular tissue excised from eyes of patients with PDR. (A‐C) Double labelling of HIF‐1α ( green ) and GFAP ( red ) with DAPI ( blue ) counterstaining in PDR fibrovascular tissues. (D‐F) Double labelling of HIF‐1α ( green ) and galctin‐1 ( red ) with DAPI ( blue ) counterstaining. (G‐I) Double labelling of HIF‐1α ( green ) and TSD22D3 ( red ) with DAPI ( blue ) counterstaining. Scale bar = 20 μm

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

doi: 10.1111/jcmm.15116

Figure Lengend Snippet: A schema showing that glucocorticoid‐transactivated TSC22D3 suppresses hypoxia‐ and diabetes‐induced galectin‐1 expression through HIF‐1α destabilization. Glucocorticoid‐bound glucocorticoid receptor (GR) transactivates TSC22D3 via glucocorticoid response element (GRE), causing ubiquitin‐proteasome system (UPS)‐mediated degradation of HIF‐1α, which is otherwise stabilized by hypoxia and diabetes for the induction of Müller glial galectin‐1 expression

Article Snippet: The protein levels of galectin‐1 in cell lysates and culture supernatants were determined with human galectin‐1 ELISA kit (R&D systems) per the manufacturer's instructions.

Techniques: Expressing