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: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 2. Hypoxia increases the expression of NR4A1 and HIF-1α and promotes glycolysis in PASMCs. (A) Cell morphology was observed by a light microscopy, scale bar = 100 μm. (B) CCK8 assays (absorbance at OD450 nm) were used to measure the cell viability of PASMCs at 0 and 24 h. (C) The levels of NR4A1 and HIF-1α were examined in PASMCs. (D) Relative mRNA levels of PKM2, GLUT1, and HK2 were examined. (E) Extracellular glucose and lactate levels were determined. n = 3. ****p < 0.0001 vs. Normoxia.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Light Microscopy

Journal: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 4. Overexpression of NR4A1 promotes glycolysis in hypoxia-induced PASMCs. (A) The effects of oe-NC and oe-NR4A1 on hypoxia- induced PASMC viability (absorbance at OD450 nm) by CCK8. (B) Relative protein expression of NR4A1 and HIF-1α determined in hypoxia- induced PASMCs. (C) The effects of NR4A1 on PKM2, GLUT1, HK2, and CD36 mRNA levels were evaluated. (D) The effects of NR4A1 on PKM2, GLUT1, HK2, and CD36 levels were measured. (E) Extracellular glucose and lactate levels were determined. n = 3. ****p < 0.0001 vs. oe-NC.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Over Expression, Expressing

Journal: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 5. NR4A1 induces HIF-1α expression to promote cell proliferation and glycolysis in hypoxia-exposed PASMCs. (A) Hypoxic PASMC viability (absorbance at OD450 nm) was measured by CCK8. (B) Protein levels of NR4A1 and HIF-1α were determined in hypoxia-induced PASMCs. (C) Relative mRNA levels of PKM2, GLUT1, HK2, and CD36 were examined in hypoxia-induced PASMCs. (D) Protein levels of PKM2, GLUT1, HK2, and CD36 were determined in hypoxia-induced PASMCs. (E) Determination of extracellular glucose and lactate levels in hypoxia-induced PASMCs. n = 3. ****p < 0.0001 vs. si-NC. ###p < 0.001, ####p < 0.0001 vs. si-NR4A1+oe-NC.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Expressing

Journal: Diabetologia

Article Title: Low levels of glucose transporters and K+ATP channels in human pancreatic beta cells early in development.

doi: 10.1007/s00125-007-0644-x

Figure Lengend Snippet: Fig. 2 Beta cell production of GLUTs, GCK and Kþ ATP channels in human fetal pancreas development. Proportions of insulin-positive cells positive for GLUT2 (black bars), GLUT1 (sloping hatched bars), GCK (horizontally hatched bars), KIR6.2 (vertically hatched bars) and SUR1 (white bars) components of Kþ ATP channels were quantified by analysis of images prepared as in Fig. 1. Results are expressed as means±SEM of pancreases of 11–15 weeks (n=5), 16–19 weeks (n=5) and 20–36 weeks (n=6) of gestation, infant pancreas (3–7.5 months of age; n=3) and juvenile and adult pancreases (n=3). mo, months; w, weeks; y, year

Article Snippet: Sections were dewaxed and rehydrated, then incubated overnight with 1:100 dilutions of primary antibodies to GLUT1, GLUT2, GCK, KIR6.2 and SUR1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques:

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Amino acid sequence identity between human GLUT1 and Japanese macaque GLUT1.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Sequencing

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Schematic showing the structure of rVSVs expressing an STLV-1 receptor molecule and AcGFP1 as a reporter protein. The gene orders in the wildtype VSV and the rVSV constructs used in this study are illustrated. The deleted G gene was replaced with the JM GLUT1 or AcGFP1 genes, yielding the VSVΔG-JmGL or VSVΔG-AcGFP constructs, respectively. In addition, a JM GLUT1 gene was inserted upstream of the AcGFP1 gene in VSVΔG-AcGFP, yielding VSVΔG-JmGL-AcGFP.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Construct

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Expression of the STLV-1 receptor molecule on the rVSV-infected target cell surface. The surface expression of JM GLUT1 protein after VSV infection was confirmed by immunofluorescence staining. VSV-permissive BHK-21 cells were infected with G-complemented VSVΔG-JmGL at an MOI of 0.1. After 1 day of culture, JM GLUT1 expressed by the viral genome was stained with anti-GLUT1-specific mouse monoclonal antibody (MAb) followed by FITC-conjugated anti-mouse IgG. The stained cells were observed by fluorescence microscopy and photographed at a constant magnification. Scale bars in panels represent 400 µm.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Infection, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Functional assessment of infectivity and specificity of rVSVs. ( A , B ) STLV-1 Env-dependent infectivity of rVSVs was evaluated. BHK-21 cells were initially mock-transfected (empty vector) or transfected with expression plasmids for STLV-1 Env derived from Si-2 cells or from STLV-1-infected JM PBMCs. After 24 h, transfected BHK-21 cells were infected with non-G-complemented rVSVs (0.25 mL each). After 3 days of culture, the cells were fixed. Subsequently, VSVΔG-JmGL-infected cells were stained with anti-GLUT1-specific MAb to detect rVSV-infected cells. On the other hand, VSVΔG-JmGL-AcGFP- or VSVΔG-AcGFP-infected cells were detected by GFP expression. Representative results from each group of rVSV-infected BHK-21 cells are shown in panel ( A ). Areas enclosed with squares are enlarged in panel ( B ). Syncytia with enlarged cell size in VSVΔG-JmGL- or VSVΔG-JmGL-AcGFP-infected cells are shown in panel ( B ). Scale bars in panels ( A , B ) represent 400 µm and 200 µm, respectively. ( C ) STLV-1 Env-dependent rVSV infection was assessed by neutralizing assay. BHK-21 cells were initially transfected with the Si-2 strain STLV-1 Env expression plasmid. After 24 h, transfected BHK-21 cells were infected with G-complemented VSVΔG-AcGFP (MOI of 0.01) or non-G-complemented rVSVs (0.25 mL each), in the absence or presence of HTLV-1 envelope glycoprotein-specific neutralizing antibody (LAT-27; 10 µg/mL). The cells infected with VSVΔG-JmGL or VSVΔG-huGL were stained and examined by VSV N-specific immunofluorescence to detect the rVSV-infected cells at 48 h post-infection. The culture of each virus without LAT-27 was defined as a control. The total number of fluorescent cells per well was determined, and the relative infectivity of each virus was calculated. The data are expressed as a percentage of the control (mean ± SD) from four independent experiments. ( D ) Viral growth kinetic assay results are shown. Si-2 strain STLV-1 Env-transfected BHK-21 cells were infected with non-G-complemented VSVΔG-AcGFP (square) or VSVΔG-JmGL-AcGFP (circle) (0.25 mL each). The total number of GFP-positive cells per well was counted to determine the quantitative viral titer (in infectious units per milliliter). The data are expressed as the mean ± SD from four independent experiments. Asterisks in panel ( C ) represent significant differences versus control (** p < 0.001 significance, by two-tailed Student’s t -test with equal variance).

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Functional Assay, Infection, Transfection, Plasmid Preparation, Expressing, Derivative Assay, Staining, Neutralizing Assay, Immunofluorescence, Virus, Control, Kinetic Assay, Two Tailed Test

Journal: Experimental and therapeutic medicine

Article Title: 3-Bromopyruvic acid regulates glucose metabolism by targeting the c-Myc/TXNIP axis and induces mitochondria-mediated apoptosis in TNBC cells.

doi: 10.3892/etm.2022.11447

Figure Lengend Snippet: Figure 4. 3‑BrPA regulates expression of c‑Myc, TXNIP and HK2. (A) Expression levels of c‑Myc, TXNIP, HK2 and GLUT1 were detected through western blot analysis. Grayscale values of the bands were determined using software. (B) 3‑BrPA downregulated c‑Myc and HK2 protein expression, whereas it upregulated TXNIP protein expression in TNBC cells (*P<0.05 vs. control group, the same cell line with 0 µM 3‑BrPA). (C) However, in non‑TNBC cells, the expression levels of c‑Myc, TXNIP, HK2 and GLUT1 were not significantly affected. The expression levels of GLUT1 were not significantly affected in both TNBC and non‑TNBC cells. TXNIP, thioredoxin‑interacting protein; HK2, hexokinase 2; GLUT1, glucose transporter 1; 3‑BrPA, 3‑bromopyruvic acid; TNBC, triple‑negative breast cancer.

Article Snippet: Then the membrane was cultured in QuickBlock Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology) at room temperature for 1 h. The membrane was incubated with antibodies against GLUT1 (1:1,000; cat. no. TA312796; OriGene Technologies, Inc.), c‐Myc (1:1,000; cat. no. TA150121; OriGene Technologies, Inc.), TXNIP (1:1,000; cat. no. TA349090; OriGene Technologies, Inc.), HK2 (1:1,000; cat. no. TA500856; OriGene Technologies, Inc.), Bcl‐2 (1:2,000; cat. no. TA806591; OriGene Technologies, Inc.), Bax (1:2,000; cat. no. TA810334; OriGene Technologies, Inc.), cytochrome c (Cyt‐C; 1:5,000; cat. no. ab13575; Abcam), Caspase‐3 (1:1,000; cat. no. TA301776; OriGene Technologies, Inc.) and β‐actin (1:1,000; cat. no. TA811000; OriGene Technologies, Inc.) at room temperature over‐ night.

Techniques: Expressing, Western Blot, Software, Control