Journal: Molecular cancer therapeutics

Article Title: BRAF inhibition decreases cellular glucose uptake in melanoma in association with reduction in cell volume

doi: 10.1158/1535-7163.MCT-15-0080

Figure Lengend Snippet: Vemurafenib induces a decrease in transmembrane glucose uptake per cell. A, 3μM Vemurafenib (+) incubation induces a decrease in transport of the non-phosphorylatable glucose analog 3-OMG normalized to cell count in sensitive lines but not B, acquired resistant lines relative to DMSO (−) after 72 hours. C, Vemurafenib treatment does not cause a significant decrease in GLUT1 or GLUT3 proteins levels in sensitive or resistant lines by 72 hours. D, Vemurafenib does not induce a significant change in GLUT1 or GLUT3 mRNA levels after 72 hours as detected by RT-qPCR.

Article Snippet: Immunoblots were conducted with the following primary antibodies all used at 1:1000: Hexokinase II (cat. no. 2867; Cell Signaling Technology, Danvers, MA, USA), Beta Actin (cat. no. 4970; Cell Signaling), Beta Tubulin (cat. no. 2128; Cell Signaling), GSK3B (cat. no. 9315), p-GSK3B S9 (cat. no. 9323; Cell Signaling), p-p90RSK T573 (cat. no. 9346; Cell Signaling), RSK1/RSK2/RSK3 (cat. no. 9355; Cell Signaling), hsp60 (cat. no. sc-1052; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GLUT1 (cat. no. 07-1401; Millipore, Billerica, MA, USA), GLUT3 (cat. no. NBP1-89762; Novus Biologicals, Littleton, CO, USA) and the secondary antibody Anti-rabbit IgG, HRP-linked Antibody (cat. no. 7074S; Cell Signaling).

Techniques: Incubation, Cell Counting, Quantitative RT-PCR

Journal: Cell Reports Methods

Article Title: In situ microwave fixation provides an instantaneous snapshot of the brain metabolome

doi: 10.1016/j.crmeth.2023.100455

Figure Lengend Snippet:

Article Snippet: GLUT3, Rabbit , Alomone , Cat# AGT-023; RRID:AB_2756644.

Techniques:

Journal: PLoS ONE

Article Title: Construction of the experimental rat model of gestational diabetes

doi: 10.1371/journal.pone.0273703

Figure Lengend Snippet: (A) Effects on the protein expression levels of GLUT1 and GLUT3 and the internal reference β-actin as determined by Western blot analysis, (B) Comparison of the protein expression levels of GLUT1 and GLUT3. * P < 0.05 vs. the NC group; # P < 0.05 vs. the HFHS group.

Article Snippet: After blocking with 5% skimmed milk in PBS-Tween, the membranes were probed with anti-glucose transporter 1 (GLUT1) or anti-glucose transporter 1 (GLUT3) primary antibody (1:500, Boeter) at 4°C overnight and conjugated with secondary antibodies at room temperature for 45 min. Antibodies against GLUT1 and GLUT3 were obtained from BOSTER Biological Technology Co., Ltd.

Techniques: Expressing, Western Blot, Comparison

Journal: Theranostics

Article Title: Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit

doi: 10.7150/thno.32915

Figure Lengend Snippet: Upregulation of Glut3 and its correlation with poor survival in CRC patients. ( A ) Gene ontology annotation shows an altered signaling pathway in primary and liver metastatic CRC. ( B ) Heatmap representation of upregulated genes associated with metabolic process. P: primary tumor; M: liver metastatic tumor. ( C and D ) High Glut3 expression was associated with lymphatic invasion, tumor grade, lymph node metastasis, tumor stage (C), and poor survival prognosis (D) in colorectal cancer patients using the TCGA dataset (COADREAD). Clinicopathological correlation was examined by a chi-square test, and survival prognosis was calculated by Kaplan-Meier analysis. ( E ) Representative IHC images of Glut3 protein expression in primary and metastatic colon cancer tissues. Scale bar=300 μm. Enlarged pictures are shown in the right panel. Unpaired t -tests were used to compare H-score analyses of primary and metastatic tumor tissues. A paired t -test was used to compare H-score analyses of paired tissue samples. ( F ) Overexpression of Glut3 was associated with poor prognosis for colon cancer (COAD) and lung squamous cell carcinoma (LUSC) in the TCGA database. HR=hazard ratio. The P value was determined by the log-rank test. ( G ) Increased glycolytic capacity in metastatic 116-LM cells. Real-time monitored glycolytic activity was determined by measuring the extracellular acidification rate (ECAR). Data are presented as the mean ± SE (left). The total cellular glycolytic capacity in 116-LM and HCT116 cells (right) is shown. ** P <0.01 versus the capacity in HCT116 cells. ( H ) Quantitative PCR analysis of glycolytic gene expression in the HCT116 and 116-LM cells. Data are representative of three independent experiments, and expression levels relative to those in HCT116 cells are shown. ( I ) Heatmap representation of cellular metabolites determined by GC/MS analysis in 116-LM and HCT116 cells. Data were obtained from two biological repeats. The glycolysis products are shown in red. ( J ) 116-LM cells were exposed to low (5 mM) or high (25 mM) concentrations of glucose for 48 h, and the expression of EMT genes was analyzed by quantitative PCR. ( K ) 116-LM and HCT116 cells (10 5 /ml) were cultured in complete medium and changed to medium with low (5 mM) or high (25 mM) concentrations of glucose for 48 h. Cell number was measured by trypan blue exclusion assay. ** P <0.01 compared to the indicated group.

Article Snippet: A pool of Glut3 lentiviral particles containing 3 target-specific shRNAs (sc-41218V) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Over Expression, Activity Assay, Real-time Polymerase Chain Reaction, Gene Expression, Gas Chromatography-Mass Spectrometry, Cell Culture, Trypan Blue Exclusion Assay

Journal: Theranostics

Article Title: Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit

doi: 10.7150/thno.32915

Figure Lengend Snippet: Glut3 promotes aggressiveness and stemness through YAP. ( A ) Quantitative PCR analysis of expression of the EMT genes in HCT116 cells stably overexpressing Glut3. ( B ) Tumor migration and invasion ability in mock- and Glut3-overexpressing HCT116 cells. Representative images are presented (left panel), and the relative percentages of migratory and invasive cells were counted (right panel). ( C and D ) Quantitative PCR analysis of the expression of stemness genes in HCT116 cells stably overexpressing Glut3 (C) or in Glut3-silenced HT29 cells (D). ( E ) Tumorsphere formation in Glut3-overexpressing HCT116 or Glut3-silenced HT29 cells. Representative images are presented (left panel), and the number of tumorspheres was counted (right panel). Scale bar=100 μm. ( F ) Luciferase reporter assay of stemness-related transcription factors in 116-LM/shRFP and 116-LM/shGlut3 cells. ( G ) Knockout of Glut3 impeded migration (a), invasion (b) and tumorsphere formation (c) in 116-LM cells. Representative images are presented (left panel), and quantitative data are shown (right panel). Scale bar=100 μm. ( H ) 116-LM and HCT116 cells were maintained in glucose-free medium for 8 h, followed by treatment with glucose for an additional 2 h. YAP expression was measured by Western blot. ( I ) 116-LM/shRFP and 116-LM/shGlut3 cells were starved with glucose deprivation or released by glucose (25 mM) treatment for an additional 2 h. Protein expression was analyzed by Western blot. ( J ) Comparison of cell invasion in Glut3-silenced HCT116 and 116-LM cells. ( K ) Quantitative PCR analysis of YAP downstream genes in Glut3-overexpressing HCT116 cells. ( L ) Knockdown of Glut3 inhibited the transcriptional activity of YAP, as measured by a TEAD luciferase reporter assay. ( M ) Knockdown of YAP abrogated tumor invasiveness and stemness in Glut3-overexpressing HCT116 cells. Representative images of migration (a), invasion (b), and tumorsphere formation (c) are presented (left panel), and quantitative data (right panel) are shown. Scale bar=100 μm. ( N ) GSEA shows that Glut3 is associated with the YAP signature. NES: net enrichment score. The results are depicted as the mean ± SEM. * P <0.05, ** P <0.01, determined by unpaired two-tailed Student's t-test.

Article Snippet: A pool of Glut3 lentiviral particles containing 3 target-specific shRNAs (sc-41218V) was obtained from Santa Cruz Biotechnology.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Migration, Luciferase, Reporter Assay, Knock-Out, Western Blot, Comparison, Knockdown, Activity Assay, Two Tailed Test

Journal: Theranostics

Article Title: Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit

doi: 10.7150/thno.32915

Figure Lengend Snippet: YAP transcriptionally regulates Glut3. ( A ) Quantitative PCR analysis of the expression of the glycolytic genes in CRC in which YAP is stably overexpressed (lower panel) or silenced (upper panel). ( B ) Illustration of three putative TEAD binding sites in the Glut3 promoter (upper panel). Chromatin immunoprecipitation analysis of YAP binding three tentative binding sites of the Glut3 promoter (lower panel). ( C ) 293T cells were cotransfected with YAP and deletion mutations of the Glut3 promoter. The transcriptional activity of Glut3 was determined by luciferase reporter assay. ( D ) Knockdown of YAP in 116-LM cells inhibited transcription of Glut3, as determined by luciferase reporter assay. ( E ) Overexpression of YAP5SA rescued Glut3 levels, and protein expression was analyzed by Western blot. ( F ) Overexpression of YAP5SA restored the capacities of 116-LM/Glut3 knockdown cells for invasiveness and stemness. Representative images of migration (a), invasion (b), and tumorsphere formation (c) are presented (upper panel), and quantitative data (lower panel) are shown. Scale bar=100 μm. The results are depicted as the mean ± SEM. * P <0.05, ** P <0.01, determined by unpaired two-tailed Student's t-test. ( G ) Pearson correlation analysis of YAP and Glut3 mRNA in the TCGA COADREAD dataset. ( H ) Kaplan-Meier curve shows that YAP and Glut3 coexpression results in a poor survival probability in colon cancer patients.

Article Snippet: A pool of Glut3 lentiviral particles containing 3 target-specific shRNAs (sc-41218V) was obtained from Santa Cruz Biotechnology.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay, Knockdown, Over Expression, Western Blot, Migration, Two Tailed Test

Journal: Theranostics

Article Title: Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit

doi: 10.7150/thno.32915

Figure Lengend Snippet: PKM2 interacts with YAP and enhances Glut3 expression. ( A ) Western blot analysis of total PKM2 and phosphorylated PKM2 levels in the whole-cell extract (WCE) and nuclear extract (NE) of HCT116 and 116-LM cells. ( B ) Western blot analysis of total PKM2 and phosphorylated PKM2 levels in the cytosolic extract (CE) and nuclear extract (NE) of 116-LM/shRFP and 116-LM/shYAP cells. ( C ) HCT116 cells were cotransfected with YAP5SA and GFP-PKM2 plasmids, and translocation of GFP-tagged PKM2 into the nucleus was detected by Western blot using an anti-GFP antibody. ( D ) Western blot analysis of PKM2 levels in 116-LM/shYAP cells in the presence of cycloheximide (CHX; 50 μg/ml). Quantification of PKM2 signals was performed using ImageJ. ( E ) Coimmunoprecipitation assay of the interaction between endogenous YAP and PKM2 in 116-LM cells (left panel). Cell lysates from HCT116 cells transfected with YAP5SA-flag and GFP-PKM2 plasmids were obtained, and coimmunoprecipitation analyses were carried out (right panel). ( F ) 116-LM cells were treated with gefitinib (10 μM) for 6 h, and cell lysates were immunoprecipitated with anti-YAP antibody, followed by immunoblotting with an anti-phospho-PKM2 antibody. The membrane was stripped and probed with an anti-PKM2 antibody. ( G and H ) Identification of the binding sites of PKM2 and YAP. Regions of interaction between PKM2 and YAP are indicated (G). Coimmunoprecipitation analyses of the deletion mutants of PKM2 and YAP5SA in HCT116 cells were carried out (H). Closed triangles denote the interaction of PKM2 with YAP, and open triangles denote the heavy chain and light chain. ( I ) HCT116 cells were transfected with YAP5SA and PKM2 plasmids, and the promoter activity of Glut3 was determined by luciferase reporter assay. ( J ) 116-LM cells were silenced with YAP, PKM2, or YAP/PKM2, and protein expression was analyzed by Western blot. ( K ) Capacities of tumor migration and invasion were determined by Transwell assays. * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed Student's t-test. Quantitative analyses of all Western blots were carried out using ImageJ software.

Article Snippet: A pool of Glut3 lentiviral particles containing 3 target-specific shRNAs (sc-41218V) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Translocation Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Membrane, Binding Assay, Activity Assay, Luciferase, Reporter Assay, Migration, Two Tailed Test, Software

Journal: Theranostics

Article Title: Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit

doi: 10.7150/thno.32915

Figure Lengend Snippet: A high-fat sucrose diet promotes tumor metastasis in a YAP-Glut3-dependent signaling axis. ( A and B ) The increased body weight (A) and fasting glucose concentration (B) of BALB/c nude mice fed a high-fat/sucrose diet (HFSD) or standard chow diet (CD). N=5 in each group. ( C ) Mice fed a high-fat/sucrose diet (HFSD) showed increased metastatic colonization of 116-LM tumors, whereas knockdown of Glut3 abrogated metastatic colonization. Representative H&E images of mouse lung sections at low- (upper panel) and high-power (lower panel) magnifications are presented. Scale bar=200 μm. The metastatic index indicates the ratio of the area of metastatic burden to the total area of lung. ( D and E ) Colon cancer cells were treated with panobinostat (0, 20, 40 nM) for 24 h. The expression of glycolytic genes was analyzed by Western blot (D), and the capacity of tumor migration and invasion was evaluated by Transwell assay (E). Quantitative analyses of all Western blots were carried out using ImageJ software. ( F and G ) NOD/SCID mice were fed an HFSD or a CD for 7 weeks followed by intraperitoneal injection with panobinostat (8 mg/kg) for an additional 6 weeks. N=4 in each group. The metastatic burden was quantified as described above (F), and the fasting glucose concentration was analyzed (G). Representative H&E images of mouse lung sections at low- (upper panel) and high-power (lower panel) magnifications are presented. Scale bar=200 μm. The results are presented as the mean ± SEM. Significant differences were analyzed by ANOVA or t -test. N.S. denotes a nonspecific difference.

Article Snippet: A pool of Glut3 lentiviral particles containing 3 target-specific shRNAs (sc-41218V) was obtained from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Knockdown, Expressing, Western Blot, Migration, Transwell Assay, Software, Injection

Journal: Neurochemistry international

Article Title: Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia.

doi: 10.1016/j.neuint.2011.06.006

Figure Lengend Snippet: Fig. 1. OGD increases GLUT expression. (A and B) The level of GLUT1 (A) or GLUT3 (B) mRNA relative to a-actin mRNA was determined by real-time PCR. Data are expressed as the mean ± S.E.M. (n = 6, different cultures). ⁄⁄p < 0.01 vs. GLUT expression in control groups (cont). (C) Western blot analysis of GLUTs and b-actin. A quantitative evaluation of GLUTs expression relative to b-actin was performed, with level in control groups taken to be 100%. Data are expressed as the mean ± S.E.M. (n = 10, different cultures). ⁄⁄p < 0.01 vs. GLUT1 expression (white bars), and ##p < 0.01 vs. GLUT3 expression (black bars) in control groups. (D) Representative immunofluorescent images. GLUT1 (upper images) or GLUT3 (lower images)-positive astrocytes (green) and cell nuclei stained with Hoechst 33342 (blue) in control groups or OGD groups are shown. Scale bars indicate 100 lm. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: When astrocytes were about 80% confluent, the CM was replaced with a penicillin/streptomycinfree CM and cultured for 1 day. siRNA Transfection Reagent mixture [about 98% siRNA Transfection Medium (Santa Cruz Biotechnology) with 0.6–0.8% GLUT3 siRNA or Control siRNA (Santa Cruz Biotechnology) supplemented with 0.8% siRNA Transfection Reagent (Santa Cruz Biotechnology)] was added to the cultures and incubated for 8 hours.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining

Journal: Neurochemistry international

Article Title: Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia.

doi: 10.1016/j.neuint.2011.06.006

Figure Lengend Snippet: Fig. 2. OGD-induced GLUTs expression is regulated through NF-jB’s signaling pathways. During OGD, wedelolactone (50 lM) or cycloheximide (100 lM) was added to the cultures and then a real-time PCR or Western blot analysis of GLUTs was performed. (A and B) Relative expression of GLUT1 (A) or GLUT3 (B) mRNA. Data are expressed as the mean ± S.E.M. (n = 4, different cultures). ⁄⁄p < 0.01 vs. GLUTs expression at 1 h of OGD (OGD1 h), and ##p < 0.01 vs. GLUTs expression at 2 h of OGD (OGD2 h). wedelo: wedelolactone treatment groups, cyclo: cycloheximide treatment groups. (C) Upper images are representative images of the control, 2 h of OGD with or without each inhibitor. Data are expressed as the mean ± S.E.M. (n = 4, different cultures). The rate of GLUTs/b-actin expression at 2 h of OGD was taken to be 100%, and used to calculate the rate of GLUTs/b-actin expression at 2 h of OGD with each inhibitor. These inhibitors significantly suppressed the increase in GLUT1 (white bars) and GLUT3 (black bars). ⁄p < 0.05, ⁄⁄p < 0.01 or #p < 0.05, ##p < 0.01 vs. 2 h of OGD. (D) Representative immunofluorescent images. GLUTs-positive astrocytes (green) and their nuclei (blue) at 2 h of OGD with or without the inhibitor. Scale bars indicate 200 lm. (E) Relative expression of NK-jB mRNA. Data are expressed as the mean ± S.E.M. (n = 6, different cultures). ⁄⁄p < 0.01 vs. control groups, and ++p < 0.01 vs. each OGD groups. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: When astrocytes were about 80% confluent, the CM was replaced with a penicillin/streptomycinfree CM and cultured for 1 day. siRNA Transfection Reagent mixture [about 98% siRNA Transfection Medium (Santa Cruz Biotechnology) with 0.6–0.8% GLUT3 siRNA or Control siRNA (Santa Cruz Biotechnology) supplemented with 0.8% siRNA Transfection Reagent (Santa Cruz Biotechnology)] was added to the cultures and incubated for 8 hours.

Techniques: Expressing, Protein-Protein interactions, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Neurochemistry international

Article Title: Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia.

doi: 10.1016/j.neuint.2011.06.006

Figure Lengend Snippet: Fig. 3. Sublethal ischemia-induced increase of GLUT3 expression contributed to the development of tolerance against subsequent lethal ischemic stress. (A) GLUT3 expression was significantly down-regulated in GLUT3 siRNA-treated astrocytes. Astrocytes were treated with scrambled (siCont) or GLUT3 siRNA (siGLUT3), and then exposed to sOGD. Representative results are shown. Lane 1; sOGD, lane 2; Control siRNA-treated astrocytes, lane 3; GLUT3 siRNA-treated astrocytes. Data are expressed as the mean ± S.E. (n = 8, different cultures). ⁄p < 0.05 vs. sOGD groups. (B) Representative GLUT3-immunofluorescent images. GLUT3-positive astrocytes (green) and their nuclei (HE, blue) in sOGD groups or GLUT3 siRNA-treated groups. Scale bar indicates 100 lm. (C) Levels of glycogen at 1 h or 1 day of reperfusion after 2 h of OGD measured with a glycogen-assay kit. The data indicate optical density values at 570 nm, and are expressed as the mean ± S.E. (n = 6, different cultures). The levels were low at 1 h of reperfusion, but were increased as compared to the control at 1 day of reperfusion. ⁄⁄p < 0.01 vs. control (Cont) groups, ##p < 0.01 vs. 1 day of reperfusion. (D) The increase in GLUT3 expression was associated with resistance to lethal OGD stress. Cell viability was determined by MTT-assay. The diagram shows the experimental protocol. The viability levels in PC (2 h of OGD)-treated groups were taken to be 100%. Data are expressed as the mean ± S.E. (n = 7, different cultures). ⁄⁄p < 0.01 vs. lethal OGD without PC, ##p < 0.01 vs. PC treatment. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: When astrocytes were about 80% confluent, the CM was replaced with a penicillin/streptomycinfree CM and cultured for 1 day. siRNA Transfection Reagent mixture [about 98% siRNA Transfection Medium (Santa Cruz Biotechnology) with 0.6–0.8% GLUT3 siRNA or Control siRNA (Santa Cruz Biotechnology) supplemented with 0.8% siRNA Transfection Reagent (Santa Cruz Biotechnology)] was added to the cultures and incubated for 8 hours.

Techniques: Expressing, Control, MTT Assay

Journal: Neurochemistry international

Article Title: Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia.

doi: 10.1016/j.neuint.2011.06.006

Figure Lengend Snippet: Fig. 4. Expression of GLUT1 was not affected by GLUT3 siRNA treatment. (A) Relative expression for a-actin of GLUT1 mRNA was performed by real-time PCR analysis. Data are expressed as the mean ± S.E.M. (n = 4, different cultures). cont: control, siGLUT3: GLUT3 siRNA-treated groups. (B) Western blot analysis of GLUT1. A quantitative evaluation of GLUTs expression for b-actin was performed, and each level in control groups was taken to be 100%. Upper images are representative blotting data of GLUT1 and b-actin. Data are expressed as the mean ± S.E.M. (n = 3, different cultures). (C) Representative GLUT1-immunofluorescent images. GLUT1-positive astrocytes (green) and their nuclei (HE, blue) in the control (cont) or GLUT3 siRNA-treated groups (siGLUT3). Scale bar indicates 100 lm. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: When astrocytes were about 80% confluent, the CM was replaced with a penicillin/streptomycinfree CM and cultured for 1 day. siRNA Transfection Reagent mixture [about 98% siRNA Transfection Medium (Santa Cruz Biotechnology) with 0.6–0.8% GLUT3 siRNA or Control siRNA (Santa Cruz Biotechnology) supplemented with 0.8% siRNA Transfection Reagent (Santa Cruz Biotechnology)] was added to the cultures and incubated for 8 hours.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot