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

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

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

doi: 10.1186/s13046-019-1393-7

Figure Lengend Snippet: Fig. 1 FAM289 was overexpressed in GBM cell lines and tissues. (A) FAM92A1 expression levels were increased in human brain cancer cells and tissues by analyzing the GBM RNA sequencing dataset from TCGA. (a) FAM92A1 expression levels were increased in human brain cancer cells compared to other kinds cancer cell lines (1. Bladder Cancer (n = 21) 2. Brain and CNS Cancer (n = 64) 3. Breast Cancer (n = 56) 4. Colorectal Cancer (n = 56) 5. Esophageal Cancer (n = 25) 6. Gastric Cancer (n = 35) 7. Head and Neck Cancer (n = 41) 8. Kidney Cancer (n = 21) 9. Leukemia (n = 83) 10. Liver Cancer (n = 29) 11. Lung Cancer (n = 166) 12. Lymphoma (n = 61) 13. Melanoma (n = 57) 14. Myeloma (n = 26) 15. Ovarian Cancer (n = 44) 16. Pancreatic Cancer (n = 44) 17. Prostate Cancer (n = 7) 18. Sarcoma (n = 39)). (b) Geometric mean of the FAM92A1 expression was significantly higher for GBM tissues compared with normal brain samples in three independent datasets. (c) Kaplan–Meier analysis of overall survival curves of FAM2A1 expression and clinical outcomes from the TCGA database. (B) The mRNA levels of FAM289 in different degrees of malignancy GBM cells lines (U251, DBTRG, U87-MG) was analyzed by western blotting (a) and qRT-PCR (b). (C) Overexpression of FAM289-GFP fusion protein in U251 cells was verified by fluorescence microscopy (a) and western blotting (b). (D) FAM289 was knocked down in U87-MG cell lines by siRNA transfection (a-c) or LentiCRISPRv2 –FAM289 infection (d-f). NABT: normal brain tissue; KD: knock down; siRNA: small interfering RNA; sgRNA: small guide RNA; Con: control; OE: overexpression; M: 100 bp DNA ladder

Article Snippet: The cells were incubated with the following antibodies for 60 min at 37 °C: rabbit anti-human FAM289 1:200 (Proteintech, USA), rabbit anti-human Galectin-1 1:200 (Proteintech, USA).

Techniques: Expressing, RNA Sequencing, Western Blot, Quantitative RT-PCR, Over Expression, Fluorescence, Microscopy, Transfection, Infection, Knockdown, Small Interfering RNA, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

doi: 10.1186/s13046-019-1393-7

Figure Lengend Snippet: Fig. 2 FAM289 is involved in GBM cells proliferation and migration in vitro and in vivo. (A) Real-time monitoring of FAM289-induced proliferation in U251 (a) or U87-MG (b) cells. Cell index was automatically recorded with the xCELLigence real-time cell analyzer (RTCA) every 15 min until the end of the experiment (60 h). Each tracing represents an average of three parallel assessments. (B) The effect of FAM289 on migrating capacity in U251 (a&b) and U87-MG (c&d) cells. The representative pictures were taken at 0 h, 24 h and 48 h after scratching. The differences were significant both at 24 h and at 48 h. (C) Transwell assay of FAM289-induced migrationin U251 (a&b) and U87-MG (c&d) cells. Images were obtained at 48 h. Data are presented as the mean ± SEM of three independent experiments. (D) Ectopic expression of FAM289 accelerated growth of FAM289 xenografts in NCG mice (n ≥5) as compared to controls. The representative tumor photos and summary volume & weight were presented in (a), (b) and (c) respectively. (E) Knockdown of FAM289 decelerated growth of U87-MG cell-derived xenografts in nude mice (n ≥5) as compared to controls (*p < 0.05 and **p < 0.01 vs. control). The representative tumor photos and summary volume & weight were presented in (a), (b) and (c) respectively. (F) The U251-Luc cells with/without overexpression FAM289 (2 × 106 each) were subcutaneously injected into the brain of NCG mice (n ≥3) and the sizes of neoplasms formed were measured with an in vivo imaging system until mice died (usually 3~4 weeks). (a) Serial pictures taken at different time points; (b) The changes of luminescence radiance values of the neoplasms formed from the injected U251-Luc cells with/ without overexpression FAM289. The difference was significant (*p < 0.05 and **p < 0.01 vs. control)

Article Snippet: The cells were incubated with the following antibodies for 60 min at 37 °C: rabbit anti-human FAM289 1:200 (Proteintech, USA), rabbit anti-human Galectin-1 1:200 (Proteintech, USA).

Techniques: Migration, In Vitro, In Vivo, Transwell Assay, Expressing, Knockdown, Derivative Assay, Control, Over Expression, Injection, In Vivo Imaging

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

doi: 10.1186/s13046-019-1393-7

Figure Lengend Snippet: Fig. 6 FAM289-mediated TMZ resistance is through stem-like property acquisition via ERK pathway. (A) The apoptosis and cell cycle of U251 and U87-MG cells treaded with TMZ were analyzed by flow cytometry. The cell apoptosis was determined by Annexin V-fluorescein isothiocyanate (FITC)/PI staining. The percentages of Annexin-V-positive cells were indicated. (B) The cell cycle was analyzed by flow cytometry after PI staining. The data were processed with ModFit LT program. (C) The in vitro colony formation in cultured U251 and U87-MG cells. Images were taken on day 10 under an inverted microscope. (D) The cell lysates from FAM289-OE U251 and FAM289-KD U87 cells were immunoblotted with anti-stem- like property factors (OCT4, c-Myc, SOX2, CD133), anti-ERK, anti-pERK and anti-DNMTs antibodies. Corresponding wild type cells were used as controls. (E) The proportion of stemness factor CD133, Ki67 and Chemokine CXCR4 expression was analyzed by flow cytometry

Article Snippet: The cells were incubated with the following antibodies for 60 min at 37 °C: rabbit anti-human FAM289 1:200 (Proteintech, USA), rabbit anti-human Galectin-1 1:200 (Proteintech, USA).

Techniques: Flow Cytometry, Staining, In Vitro, Cell Culture, Inverted Microscopy, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

doi: 10.1186/s13046-019-1393-7

Figure Lengend Snippet: Fig. 7 High FAM289 expression in GBM tissues correlated with poor prognosis. (A) Representative images of FAM289, galectin-1 and Ki67 protein expression in GBM tumor tissues and normal brain tissue by immunohistochemistry. (B) FAM289 and Galectin-1 protein in fresh-frozen specimens from normal human brain and GBM tissues was detected by western blotting. GAPDH was used as a loading control. *P < 0.05 vs control. (C) Kaplan–Meier analysis of overall survival curves of in early-stage (I–II) and advanced-stage (III–IV) GBM patients with high FAM289 expression versus low FAM289 expression

Article Snippet: The cells were incubated with the following antibodies for 60 min at 37 °C: rabbit anti-human FAM289 1:200 (Proteintech, USA), rabbit anti-human Galectin-1 1:200 (Proteintech, USA).

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

doi: 10.1186/s13046-019-1393-7

Figure Lengend Snippet: Fig. 8 Schematic representation of the proposed mechanism of FAM289 in GBM cells. FAM289 contributes to tumorigenesis in malignant glioma by interacting with Galectin-1 to promote FAM289 protein translocation into cell nucleus, which could activate the ERK pathway to up regulate DNMTs expression and increase stem-like property gene expression, thereby affecting its drug sensitivity of TMZ in the treatment of glioma. Gal-1: Galectin-1, β1: β1 integrin, p: phosphorylation

Article Snippet: The cells were incubated with the following antibodies for 60 min at 37 °C: rabbit anti-human FAM289 1:200 (Proteintech, USA), rabbit anti-human Galectin-1 1:200 (Proteintech, USA).

Techniques: Translocation Assay, Expressing, Gene Expression, Phospho-proteomics