Journal: World Journal of Surgical Oncology

Article Title: LAIR-1 overexpression inhibits epithelial–mesenchymal transition in osteosarcoma via GLUT1-related energy metabolism

doi: 10.1186/s12957-020-01896-7

Figure Lengend Snippet: LAIR-1 inhibits Glut1-related glucose uptake in OS cells. a Heatmap showing the levels of differentially expressed mRNAs. b Top 20 KEGG pathway annotation categories for target gene functions of predicted mRNAs. c Selected significantly differentially expressed mRNA-related to EMT in RNA-seq data between two groups, *** P < 0.001. d qPCR validation of differentially expressed EMT-related genes in LV-NC and LV-LAIR-1-overexpressing OS cells, ** P < 0.01. e Glut1 expression analyzed by western blotting. f Immunofluorescence staining of Glut1 in the LV-LAIR-1-overexpressing OS cells. Scale bar = 50 μm. Data were obtained from at least two independent experiments.

Article Snippet: Total protein was extracted using a routine procedure and blotted with the following primary antibodies: LAIR-1 (sc-398141; Santa Cruz Biotechnology), phospho-Foxo1 (Ser256) (84192; Cell Signaling Technology, Danvers, MA, USA), Foxo1 (2880; Cell Signaling Technology), phospho-Akt (Ser473) (AF8355; Affinity Biosciences, Cincinnati, OH, USA), Akt (9272; Cell Signaling Technology), proliferating cell nuclear antigen (PCNA; BM0104; Boster Biotech Co., Ltd., Wuhan, China), Twist1 (ab50581; Abcam, Cambridge, UK), Glut1 (NB110-39113, Novus Biologicals, Littleton, CO, USA), and β-actin (30101ES50; Yeasen Biotech Co., Ltd., Shanghai, China).

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining

Journal: OncoImmunology

Article Title: Neutrophil extracellular traps induce tumor metastasis through dual effects on cancer and endothelial cells

doi: 10.1080/2162402x.2022.2052418

Figure Lengend Snippet: Figure 3. Glycolytic activation mediates the release of NETs by tumor neutrophils. (a) CD15+ cells were purified from paired non-tumor and tumor tissues of HCC patients, and their levels of Cit-H3 and GLUT1 expression were quantified by western blotting (n = 3). (b-d) CD15+ cells were purified from tumor tissues of HCC patients. The levels of and correlations between Cit-H3 and GLUT1 expression in these cells were analyzed by flow cytometry (n = 6 in b, c; n = 14 in d). (e) Paraffin-embedded HCC samples were stained with anti-human CD15 antibody (red), anti-human Cit-H3 antibody (white), anti-human GLUT1 antibody (green), and DAPI (blue). The co- expression of Cit-H3 and GLUT1 on CD15+ tumor-infiltrating neutrophils was analyzed by confocal microscopy (n = 3). (f, g) CD15+ cells were purified from peripheral blood of healthy donors and treated with 30% primary HCC cancer cell supernatants (T-SN) or paired non-tumor liver cell supernatants (N-SN) for 12 hours, in the presence or absence of glycolytic inhibitor 2DG or G6PD inhibitor 6AN. These cells were then stained with anti-human Cit-H3 antibody (green), and their release of DNA and levels of Cit-H3 expression were visualized and evaluated by confocal microscopy (n = 3). Blue: DAPI. Results shown in c, f, and g are expressed as the means ± SEMs. The following statistical analyses were performed: Student’s t-test (c), Pearson correlation and linear regression analysis (d), one-way ANOVA with Bonferroni posttest (f, g). * P < .05, ** P < .01, *** P < .001.

Article Snippet: Leukocytes were stained with fluorescent-conjugated antibodies anti-human CD15 (48–0158, eBioscience, San Diego, U.S.), anti-human GLUT1 (FAB1418A, R&D Systems, Minneapolis, U.S.), anti-human CD45 (A96416, Beckman Coulter, Brea, U.S.), or non-fluorescent-conjugated antihuman Cit-H3 antibody, and subjected to direct or indirect flow cytometry analysis.

Techniques: Activation Assay, Purification, Expressing, Western Blot, Flow Cytometry, Staining, Confocal Microscopy

Journal: OncoImmunology

Article Title: Neutrophil extracellular traps induce tumor metastasis through dual effects on cancer and endothelial cells

doi: 10.1080/2162402x.2022.2052418

Figure Lengend Snippet: Figure 4. Glycolytic activation induced the release of NETs via NOX-ROS pathway. CD15+ cells were purified from peripheral blood of healthy donors and treated with 30% primary HCC cancer cell supernatants (T-SN) or paired non-tumor liver cell supernatants (N-SN) for 12 hours, in the presence or absence of 2DG, or 6AN, or DPI. (a) The enzyme activity of NOX in these cells was quantified (n = 3). (b-d) Levels of ROS production by these cells were evaluated by flow cytometry (n = 6). (e, f) Levels of Cit-H3 expression in these cells were analyzed by confocal microscopy (n = 6). Green: Cit-H3; Blue: DAPI. (g, h) CD15+ cells were purified from tumor tissues of HCC patients. Correlations between levels of ROS, GLUT1, and Cit-H3 expression in these cells were determined by flow cytometry (n = 12). Results shown in a, c, d, and f are expressed as the means ± SEMs. The following statistical analyses were performed: Student’s t-test (a), Kruskal–Wallis test followed by Dunn’s posttest (c, d), one-way ANOVA with Bonferroni posttest (f), Pearson correlation and linear regression analysis (g, h). * P < .05, ** P < .01, *** P < .001.

Article Snippet: Leukocytes were stained with fluorescent-conjugated antibodies anti-human CD15 (48–0158, eBioscience, San Diego, U.S.), anti-human GLUT1 (FAB1418A, R&D Systems, Minneapolis, U.S.), anti-human CD45 (A96416, Beckman Coulter, Brea, U.S.), or non-fluorescent-conjugated antihuman Cit-H3 antibody, and subjected to direct or indirect flow cytometry analysis.

Techniques: Activation Assay, Purification, Activity Assay, Flow Cytometry, Expressing, Confocal Microscopy