Journal: Autophagy

Article Title: Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.

doi: 10.1080/15548627.2023.2239042

Figure Lengend Snippet: Figure 6. Periplocin binds and prevents ubiquitin-mediated degradation of LGALS3 in CRC cells. (A) Immunoblotting analysis of LGALS3 in cells treated with periplocin for 24 h at the indicated concentrations. (B and C) Representative images (B) and quantitative analysis (C) of immunohistochemical staining for LGALS3 in SW480 ×enografts from vehicle- or periplocin-treated mice. Scale bar: 50 μm. (D) Immunoblotting analysis of LGALS3 in cells treated with 0.50 μM periplocin for 24 h in the presence or absence of cycloheximide (CHX, 50 μg/mL). (E) Quantitation of LGALS3 protein level in (D). (F) Immunoblotting analysis of LGALS3 in cells treated with 0.50 μM periplocin for 24 h in the presence or absence of MG132 (25 μM, 6 h). (G) Quantitation of LGALS3 protein level in (F). (H) FLAG-LGALS3 was co-expressed

Article Snippet: LGALS3 (MAB1154) was purchased from R&D Systems.

Techniques: Ubiquitin Proteomics, Western Blot, Immunohistochemical staining, Staining, Quantitation Assay

Journal: Autophagy

Article Title: Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.

doi: 10.1080/15548627.2023.2239042

Figure Lengend Snippet: Figure 7. Periplocin induces lethal lysophagy by upregulating LGALS3 in CRC cells. (A and B) Immunoblotting analysis of LC3B turnover in cells transfected with siNC or siLGALS3 followed by 0.50 μM periplocin treatment for 24 h. (C and D) Immunoblotting analysis of LC3B turnover in parental or lgals3 KO cells followed by 0.50 μM periplocin treatment for 24 h. (E and F) Representative images (E) and quantitative analysis (F) for immunofluorescent staining of endogenous LC3B puncta in cells transfected with siNC or siLGALS3 followed by 0.50 μM periplocin treatment for 24 h. Scale bar: 10 μm. (G and H) MTT assay of CRC cells with or without LGALS3 knockout (G, lgals3 KO#1; H, lgals3 KO#2) in response to 0.50 μM periplocin treatment for 24 h. (I) Colony formation assay of parental or lgals3 KO cells treated with or without 0.50 μM periplocin for 24 h. (J) Quantification of clone numbers in (I). (K and L) DLD-1 parental or lgals3 KO cells were subcutaneously inoculated into nude mice. Mice were injected with vehicle or periplocin (15 mg/kg/day) for two weeks. Image (K) and weight (L) of tumor xenografts were shown. Results in A-J are representative of three independent experiments. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.

Article Snippet: LGALS3 (MAB1154) was purchased from R&D Systems.

Techniques: Western Blot, Transfection, Staining, MTT Assay, Knock-Out, Colony Assay, Injection

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: The Journal of Clinical Investigation

Article Title: ADAMTS7 promotes smooth muscle foam cell expansion in atherosclerosis

doi: 10.1172/JCI187451

Figure Lengend Snippet: ( A ) Schematic outlining experimental design and the generation of the SMC transgenic ADAMTS7 mouse. Created with BioRender.com. ( B ) ORO staining of the en face aorta and quantification of lesion area. n = 9. ( C ) Representative images of H&E-stained aortic root sections, quantification of plaque areas ( n = 6). Scale bar: 500 μm. ( D ) Representative images of H&E-stained aortic root sections with necrotic core outlined in dotted line ( n = 6 mice). Scale bar: 300 μm. ( E ) Representative images of aortic root sections stained against α-SMA (Cy3; red), MAC2 (Alexa Fluor 488 green), and cell nuclei (DAPI blue) with their subsequent quantification relative to lesion area ( n = 6). Scale bar: 300 μm. ( F ) Representative Picrosirius red staining of aortic root lesions with bars indicating fibrous cap thickness and quantification of fibrous cap thickness ( n = 6). Scale bar: 300 μm. ( G ) Schematic outlining experimental design and the generation of the EC transgenic ADAMTS7 mouse. Created with BioRender.com. ( H ) ORO staining of the en face aorta and quantification of lesion area. n = 3–4. ( I ) Representative images of H&E-stained aortic root sections and quantification of plaque areas ( n = 5–6). Scale bar: 500 μm. **** P < 0.0001, * P < 0.05. Statistics were analyzed using a 2-tailed Student’s t test.

Article Snippet: The sections then were stained using the following Abs and dilutions: 1:1,000 α-SMA-Cy3 (MilliporeSigma, C6198) and 1:500 MAC2 (Cedarlane, CL8942AP).

Techniques: Transgenic Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

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