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: 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

Journal: International Journal of Biological Sciences

Article Title: Delactylase effects of SIRT3 on a positive feedback loop involving the RUNX1-glycolysis-histone lactylation in diabetic kidney disease

doi: 10.7150/ijbs.126011

Figure Lengend Snippet: Knockdown of RUNX1 inhibits TECs glycolysis and ameliorates renal fibrosis in DKD. A. Schematic illustration of AAV9-shRUNX1 injection in the db/db group; B-C. Blood glucose levels Urinary albumin creatinine ratio (UACR) in db/m, db/db, and db/db + shRUNX1 group (n=6); D. Representative images of PAS, Masson, Sirius red staining and IHC staining (NF-κB p65, αSMA and FN) (scale bar=50 μm) in db/m, db/db, and db/db + shRUNX1groups (n=6); E-F. Protein levels of RUNX1, αSMA and FN in db/m, db/db, and db/db + shRUNX1 groups by western blot, with semi-quantitative analyses (n=6); G. The lactate concentration in HK-2 cells in the Control, HG and HG + siRUNX1 group (n=6); H. The ECAR levels after culturing with glucose followed by oligomycin and 2-DG in Control, HG and HG + siRUNX1 group (n=3); I. Protein levels of HK1 and SLC2A1 in Control, HG and HG + siRUNX1 group (n=3). Data represent the mean ± SD from three independent experiments. # P <0.05 versus db/m, and ** P <.01 versus db/db group or HG by one-way or two-way ANOVA.

Article Snippet: Primary antibodies targeting E-cadherin (sc-59778, Santa Cruz Biotechnology), PKM2(NBP1-48308, Novus Biologicals) H4K12la (PTM-1411RM, PTMBio), RUNX1 (sc-365644, Santa Cruz Biotechnology), HK1 (ab150423, Abcam), SLC2A1 (MAB1418, R&D Systems), FN (sc-8422, Santa Cruz Biotechnology), αSMA (NB300-978, Novus Biologicals) then incubated with the secondary antibodies for one hour.

Techniques: Knockdown, Injection, Staining, Immunohistochemistry, Western Blot, Concentration Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Delactylase effects of SIRT3 on a positive feedback loop involving the RUNX1-glycolysis-histone lactylation in diabetic kidney disease

doi: 10.7150/ijbs.126011

Figure Lengend Snippet: Lactate-mediated elevation of H4K12la initiates RUNX1/glycolysis pathway. A. The lactate concentration in HK-2 cells in the Control, HG and HG + FX-11 group (n=6); B-C. Protein levels of RUNX1 in Control, HG and HG + FX-11 group in HK-2 cells by western blot, with semi-quantitative analyses (n=3); D. The mRNA level of RUNX1 in the Control, HG and HG + FX-11 group in HK-2 cells by RT-qPCR (n=5); E. H4K12la occupancy analysis of RUNX1 in the Control, HG and HG + FX-11 group in HK-2 cells by CUT&Tag-qPCR (n=3); F. Immunostained for H4K12la (yellow), RUNX1 (red) and E-cadherin (green), counterstained with DAPI (blue) in db/m, db/db, db/db + FX-11 groups (scale bar=50 μm); G. Double immunostained RUNX1 (red) and H4K12la (green), and counterstained with DAPI (blue) by IF staining in HK-2 cells in Control, HG and HG + FX-11 group, scale bar=50 μm (n=3); H. ATP content and glucose consumption in Control, HG, HG + FX-11 and HG + FX-11 + RUNX1 group in HK-2 cells (n=6); I. Mitochondrial oxidative capacity in HK-2 cells in the Control, HG, HG + FX-11 and HG + FX-11 + RUNX1 group (n=3); J. The ECAR levels after culturing with glucose followed by oligomycin and 2-DG in Control, HG, HG + FX-11 and HG + FX-11 + RUNX1 group (n=3); K-L. Protein levels of HK1 and SLC2A1 in HK-2 cells in the Control, HG, HG + FX-11 and HG + FX-11 + RUNX1 group by western blot, with semi-quantitative analyses (n=3); M. Double immunostained SLC2A1 (red) and HK1 (green), and counterstained with DAPI (blue) by IF staining in HK-2 cells in HG, HG + FX-11 and HG + FX-11 + RUNX1 group, scale bar=50 μm (n=3). Data represent the mean ± SD from three independent experiments. ** P <.01 versus HG group (A, C, D, E, H) or HG + FX-11 group (H, I, J, L) by one-way ANOVA.

Article Snippet: Primary antibodies targeting E-cadherin (sc-59778, Santa Cruz Biotechnology), PKM2(NBP1-48308, Novus Biologicals) H4K12la (PTM-1411RM, PTMBio), RUNX1 (sc-365644, Santa Cruz Biotechnology), HK1 (ab150423, Abcam), SLC2A1 (MAB1418, R&D Systems), FN (sc-8422, Santa Cruz Biotechnology), αSMA (NB300-978, Novus Biologicals) then incubated with the secondary antibodies for one hour.

Techniques: Concentration Assay, Control, Western Blot, Quantitative RT-PCR, Staining

Journal: International Journal of Biological Sciences

Article Title: Delactylase effects of SIRT3 on a positive feedback loop involving the RUNX1-glycolysis-histone lactylation in diabetic kidney disease

doi: 10.7150/ijbs.126011

Figure Lengend Snippet: SIRT3 increased RUNX1 mediated glycolysis and promotes renal fibrosis in DKD. A-B. Protein levels of SIRTS, RUNX1 and αSMA in db/m, db/db, and db/db + SIRT3 groups by western blot, with semi-quantitative analyses (n=6); C. Representative images of Masson, Sirius red staining and IHC staining (RUNX1, NF-κB p65, αSMA and FN) (scale bar=50 μm) in db/m, db/db, and db/db + sSIRT3 groups (n=6); D. Protein level of HK1, SLC2A1, FN and αSMA by western blot in the HG, HG + SIRT3 and HG + SIRT3 + RUNX1 group in HK-2 cells, and its semi-quantitative analysis (n=3); E. Double immunostained αSMA (red) and RUNX1 (green), and counterstained with DAPI (blue) by IF staining in HK-2 cells in HG, HG + SIRT3 and HG +SIRT3 + RUNX1 group, scale bar=50 μm (n=3). Data represent the mean ± SD from three independent experiments. * P <.05 or ** P <.01 versus db/db group (B) or HG +SIRT3 group (D) by one-way ANOVA.

Article Snippet: Primary antibodies targeting E-cadherin (sc-59778, Santa Cruz Biotechnology), PKM2(NBP1-48308, Novus Biologicals) H4K12la (PTM-1411RM, PTMBio), RUNX1 (sc-365644, Santa Cruz Biotechnology), HK1 (ab150423, Abcam), SLC2A1 (MAB1418, R&D Systems), FN (sc-8422, Santa Cruz Biotechnology), αSMA (NB300-978, Novus Biologicals) then incubated with the secondary antibodies for one hour.

Techniques: Western Blot, Staining, Immunohistochemistry