Journal: The Journal of Biological Chemistry

Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

doi: 10.1074/jbc.M109.073924

Figure Lengend Snippet: Minimal hypoxia induction of Ror2 expression. A, Ror2 expression is minimally induced after 48 h of exposure to cobalt chloride. Normoxic (N) and CoCl2 (C)-treated protein samples were immunoblotted with HIF-2α and Egln3 antibodies to show that HIF-2α and the HIF target Egln3 were induced in VHL(+) cell lines. Ror2 levels remained stable to this manipulation. Ku80 antibody was used as a loading control (LC). B, left, ROR2 mRNA levels are minimally induced under hypoxic-like conditions in RCC4 cells. qRT-PCR analysis of the hypoxia mimetic cobalt chloride (CoCl2) transcriptional induction after 24 h in the VHL expressing cell line RCC4 3-14 demonstrate induction of the HIF target gene EGLN3 (**, p = 0.0015) in response to treatment with hypoxia mimetic, confirming HIF transcriptional activity. Ror2 mRNA was not significantly induced under these conditions. Right, Ror2 mRNA levels are minimally induced under hypoxic-like conditions in 786-0 WT8 cells. qRT-PCR analysis of the hypoxia mimetic DMOG transcriptional induction after 24 h in the VHL expressing cell line 786-0 WT8 demonstrate induction of HIF target genes EGLN3 (**, p = 0.0091) and GLUT1 (**, p = 0.0015) in response to treatment with the hypoxia mimetic, confirming HIF transcriptional activity. Ror2 transcript levels show minimal enrichment upon treatment. Transcript values are shown as normalized to the β-actin RNA internal standard and relative to the unstimulated cells of each set of paired cells. Error bars represent ± S.E.

Article Snippet: The HIF-1α antibody was obtained from BD Transductions (Franklin Lakes, NJ) and the Glut1 and Egln3 antibodies were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 2. Effect of hypoglycemia, normoglycemia and hyperglycemia on the level of GLUT1 in plasma membrane in normoxic and hypoxic conditions in FTC-133 and 8305c cells. GLUT1, glucose transporter 1.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Clinical Proteomics, Membrane

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 1. Effect of hypoglycemia, normoglycemia and hyperglycemia on the expression of GLUT1, GLUT3 and HIF1α in normoxic and hypoxic condi- tions in FTC-133 (A) and 8305c cells (B). Differences in GLUT1 and HIF1α expression levels in FTC-133 and 8305c cells (C). The immunodetection of GLUT1 and HIF1α in lysates from the cells treated with CoCl2 was performed on one blot to show the expression differences. GLUT1, glucose transporter 1; HIF1α, hypoxia inducible factor α.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Expressing, Immunodetection

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 3. (A) Reduced expression of GLUT1 in FTC-133 and 8305c cells growing in hyperglycemia is correlated with reduced phosphorylation of Ser473 AKT1. (B) The intensity of the bands was analyzed by densitometry. GLUT1, glucose transporter 1.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Expressing, Phospho-proteomics

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 6. (A) GLUT1 mRNA, (B) total protein and (C) membrane expression levels in FTC-133 and 8305c cells 48 or 72 h after siRNA treatment. GLUT1, glucose transporter 1.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Membrane, Expressing

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 7. Effect of GLUT1 expression downregulation on (A) FTC-133 and (B) 8305c cell viability. Data are presented as the average of at least three independent experiments performed in triplicate (± SD) *P<0.05, **P<0.01 and ***P<0.001. GLUT1, glucose transporter 1.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Expressing

Journal: Oncology reports

Article Title: Glucose-dependent glucose transporter 1 expression and its impact on viability of thyroid cancer cells.

doi: 10.3892/or.2014.3673

Figure Lengend Snippet: Figure 8. Effect of GLUT1 expression downregulation on glucose analog 2-NBDG uptake to (A) FTC-133 and (B) 8305c cells. Data are presented as the average of at least three independent experiments performed in tripli- cate (± SD) *P<0.05, **P<0.01 and ***P<0.001. GLUT1, glucose transporter 1.

Article Snippet: The antibodies from Correspondence to: dr anna krześlak, department of Cytobiochemistry, Faculty of Biology and environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland E-mail: krzeslak@biol.uni.lodz.pl Key words: GLUT1, HIF1α, AKT1, glucose uptake, cell viability santa Cruz Biotechnology, Inc., (santa Cruz, Ca, Usa) used were: mouse monoclonal anti-GLUT3, rabbit polyclonal anti-HIF1α, mouse monoclonal anti-β-actin, goat polyclonal anti-rabbit IgG-HRP and goat polyclonal anti-mouse IgG-HRP.

Techniques: Expressing

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