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: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 2. The GLUT3 protein is upregulated in hypoxic immortalized prostate epithelial cells. (Upper and Middle panels) RWPE1 cells were cultured in either normoxic or under chronic hypoxic (1% O2) conditions for 48 h and then processed for immunofluorescence microscopy. Cells were stained for either CA9, GLUT1, or GLUT3 (green, Upper and Middle panels). DNA, blue. Scale, 25 µm. (Lower panels) Comparison of flow cytometry profiles of CA9, GLUT1, or GLUT3 in cells cultured in normoxia (red) versus hypoxia (yellow). Cells stained with only secondary antibody is shown (dark red). The fold increase in level of the indicated proteins is denoted. PE, phycoerythrin.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Cell Culture, Microscopy, Staining, Comparison, Cytometry

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 3. GLUT3 co-localizes with pimonidazole (Hypoxyprobe) in mouse xenograft tumors formed from prostate epithelial CTN-2-2 cells. Non-tumorigenic immortalized prostate epithelial PrEC-Hahn cells were transformed by transiently inducing chromosomal instability. The resulting tumorigenic line was named CTN-9, which produced malignant solid xenograft tumors in subcutaneously in- jected NSG mice [52]. Cell lines isolated from these tumors were named CTN1-2 and 2-2 lines [52]. (Upper panel) Single tumor section stained for pimonidazole (PIMO) (green) and GLUT3 (red). DNA, blue. Boxed regions are shown at higher magnification in the lower panels. (Lower panels) Higher magnification images from boxed regions 1 or 2 in upper panel. Serial tissue sections were stained for hematoxylin and eosin (H&E), PIMO (green), and GLUT3, GLUT1 or CA9 (red) as indicated. Concentrations of GLUT3 (yellow arrows) and CA9 (white arrows) that juxtapose with PIMO are marked. Dashed white boxes show GLUT3 concentrated at the plasma membrane in tumor cells (insets). Scale, 100 µm.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Transformation Assay, Produced, Isolation, Staining, Clinical Proteomics, Membrane

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 4. GLUT3 co-localizes with pimonidazole (Hypoxyprobe) in mouse xenograft tumors formed from prostate cancer DU145 cells. (Upper panel) Single tumor section stained for pimonidazole (PIMO) (green) and GLUT3 (red). DNA, blue. Boxed regions are shown at higher magnification in the lower panels. (Lower panels) Higher magnification images from boxed regions 1, 2, or 3 in upper panel. Serial tissue sections were stained for hematoxylin and eosin (H&E), PIMO (green), and GLUT3, GLUT1 or CA9 (red) as indicated. Concentrations of GLUT3 (yellow arrows) and CA9 (white arrows) that juxtapose with PIMO are marked. Dashed white boxes show GLUT3 concentrated at the plasma membrane in tumor cells (insets). Scale, 100 µm.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Staining, Clinical Proteomics, Membrane

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 5. Measurements of the co-localization of hypoxia markers in xenograft tumors show a high degree of association between pimonidazole and GLUT3. (a) Single section from a CTN2-2 tumor immunostained for pimonidazole (PIMO) (green) and GLUT3 (red). DNA, blue. Scale, 100 µm. (b) To quantify co-localization of hypoxia markers GLUT1, GLUT3, and CA9 with PIMO, binary tracings were placed around the PIMO signal (green lines) defined a threshold of intensity specific to each tumor. Binaries were then dilated >0–15 µm (orange lines) and >15–30 µm (red lines) to capture the adjacent signal of each hypoxia marker. Same image as in (a). (c) Graphs show fold change in mean fluorescence intensities of hypoxia markers GLUT1, GLUT3 and CA9 within three PIMO-positive or adjacent regions captured within the expanded binary masks: 0 µm where the PIMO signal resides, >0–15 µm (orange bars) and >15–30 µm (red bars). Mean fluorescence intensities within these three regions was divided by ‘background’ signal from regions >30 µm from the PIMO signal. A dashed line indicates no change in signal relative to the background. Four optical fields in tissue sections from two different DU145, CTN1-2 and CTN2-2 tumors were analyzed (six different tumors examined in total).

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Marker

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 6. Patient-derived xenograph (PDX) model of primary prostate cancer contains distinct pockets of GLUT3 staining. (a) Patient-derived xenograft (PDX) from a primary prostate tumor stained with H&E. Scale, 1.5 mm. (b) Section of PDX tumor immunostained for E-Cadherin (green) to mark cell borders and GLUT3 (red). DNA, blue. Boxed region is shown at higher magnification in (c). Scale, 250 µm. (c) Serial tissue sections were immunostained for either GLUT3 (red, left panel), a cocktail containing anti-GLUT1 and anti-CA9 antibodes (red, right panel), or antibodies against all three proteins GLUT1, GLUT3, and CA9 (red, middle panel) as indicated. Dashed white boxes show GLUT3 concentrated at the plasma membrane in tumor cells (insets). Scale, 100 µm.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Derivative Assay, Staining, Clinical Proteomics, Membrane

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 7. Patient-derived xenograph (PDX) model of bone metastatic prostate cancer contains distinct pockets of GLUT3 staining. (Upper panel) Section of PDX tumor immunostained for E-Cadherin (green) to mark cell borders and GLUT3 (red). DNA, blue. Boxed regions are shown at higher magnification (Lower panels). Scale, 500 µm. (Lower panels) Higher magnification images from boxed regions 1 or 2 in upper panel. Serial tissue sections were stained with H&E (column 1) or immunostained for either GLUT3 (red, column 2), a cocktail containing anti-GLUT1, GLUT3, and CA9, antibodies (red, column 3), or antibodies against GLUT1 and CA9 (red, column 4) as indicated. Dashed white boxes show GLUT3 concentrated at the plasma membrane in tumor cells (insets). Scale, 100 µm.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Derivative Assay, Staining, Clinical Proteomics, Membrane

Journal: Diagnostics (Basel, Switzerland)

Article Title: GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

doi: 10.3390/diagnostics12030676

Figure Lengend Snippet: Figure 8. Compared to hypoxia biomarkers GLUT1 and CA9, GLUT3 labels larger areas within PDX tumor sections. (a) Patient-derived xenograft (PDX) from a primary prostate tumor immunostained with the triple anti-CA9, GLUT1, and GLUT3 antibody cocktail. Scale, 100 µm. (b) Same image as in (a) with binary mask to outline regions of the triple stain. (c) Graph shows measurements of total area of each stain within 3 different PDX prostate tumors. Three sequential sections were obtained from each tumor, stained for either CA9/GLUT1, GLUT3, or the triple stain CA9/GLUT1/GLUT3, and imaged. Total area of staining was quantified by masking regions of high intensity staining using Nikon Elements image analysis software. Note, in the metastatic prostate PDX tumor samples, the vast majority of the signal observed in the triple stain is due to the GLUT3 stain. However, in the primary prostate PDX, GLUT3 staining alone is not as effective at revealing putative hypoxic regions compared to the triple stain.

Article Snippet: Antibodies used include GLUT1 (Atlas, Fort Collins, CO, USA, HPA058494), GLUT3 (Proteintech, Rosemont, IL, USA, 20403-IAP), and CA-9 (Novus, Littleton, CO, USA, NB100-417SS) at 1:1000 dilution.

Techniques: Derivative Assay, Staining, Software

Journal: Alcohol and alcoholism (Oxford, Oxfordshire)

Article Title: Glycosylation is altered by ethanol in rat hippocampal cultured neurons.

doi: 10.1093/alcalc/agl044

Figure Lengend Snippet: Fig. 4. Western blot analysis of the glucose transporters GLUT1 and GLUT3 in control (CNT) and alcohol-exposed (EtOH) neurons. Histograms show increases in densitometric readings of GLUT1 and GLUT3 in alcohol-exposed cells compared with the controls. The data used for the statistical analysis are the mean ± SD of four independent experiments. Asterisks indicate significant differences (Student’s t-test P < 0.05).

Article Snippet: Antibodies: anti-MAP2 mouse monoclonal antibody (Sigma-Aldrich, Spain); anti-neurofilament (NF) 200 rabbit polyclonal antibody (Sigma-Aldrich); anti-GLUT1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc. CA, USA), anti-GLUT3 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), anti-digoxigenin (DIG) mouse monoclonal antibody (Roche Diagnostics, S.L.

Techniques: Western Blot, Control