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: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Gene expression level (RSEM) of glucose transporters in TCGA-LUAD samples (n = 511). ( b ) Immunohistochemistry (IHC) from a next-generation tissue microarray of human LUAD and LUSC showing score 1 (intermediate) or score 2 (strong) GLUT1 staining. The number of cases per score and histology are indicated. Scale bars: 100 μm. ( c ) Expression level (log2 normalized) of SLC2A1 and SLC2A3 in the 4 NMF LUAD subtypes. Percent of TP53 mutation in each subtype is indicated. ( d ) Graph with mean ± s.e.m. shows the fold changes of KP and KPG1 tumor volumes (n = 32 and 26, respectively) monitored during 28 days by μCT, starting at 16 weeks and 6 days post-tumor initiation with tumor volumes set to 1. ns: not significant by Mann-Whitney test. ( e ) Dot plot with mean ± s.d. shows KP and KPG1 tumor weights (n = 17 and 21) at sacrifice 29 weeks post-tumor initiation. ns: not significant by Mann-Whitney test. ( f ) Dot plot with mean ± s.d. shows the average number of KP and KPG1 tumors per mouse (n = 7 and 6 mice). ns: not significant by Mann-Whitney test. ( g ) Percent of KP (n = 128) and KPG1 (n = 102) lesions classified by tumor grades, either detailed from alveolar hyperplasia (AH) to grade 5 or discriminated between alveolar hyperplasia and adenomas, and adenocarcinomas. Alveolar hyperplasia and adenomas include the AH and the tumor grades 1, 2, and 3. Adenocarcinomas contain the tumor grades 4 and 5. **: p < 0.01. Fisher test was applied when comparing AH, grade 1, grade 2, grade 3, grade 4, and grade 5. Chi-square for trend was applied when comparing alveolar hyperplasia and adenomas, and adenocarcinomas. ( h ) Kaplan-Meier survival analysis of KP (n = 8) and KPG1 (n = 6) mice. ns: not significant by Log-rank test. Figure 1—source data 1. Source files for tumor growth, grades and survival of KP and KPG1 mice.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Gene Expression, Immunohistochemistry, Microarray, Staining, Expressing, Mutagenesis, MANN-WHITNEY

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Kaplan-Meier survival analyses of glucose transporters in TCGA-LUAD (n = 448). Samples are split by median expression into high and low groups. p-values were computed with Wald test. ( b ) Heatmap showing gene expression (log2 normalized) of 935 metagenes selected by NMF to identify the 4 LUAD subtypes: NMF1-4 (top bar 1). Top bars 2 and 3: SLC2A1 and SLC2A3 expression, respectively. Top bar 4: TP53 mutation status. Top bar 5: samples identified as proximal proliferative, proximal inflammatory and terminal respiratory unit . ( c ) NMF subtype prediction applied to KP mouse bulk Lenti tumors; NMF1: 8%, NMF2: 26%, NMF3: 18% and NMF4: 48% (average of 4 samples).

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Expressing, Gene Expression, Mutagenesis

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) (upper panels) Representative examples of Glut1 staining by immunohistochemistry (IHC) in KP tumors showing weak, intermediate or strong expression. Scale bars: 100 μm. (lower panel) IHC quantification of KP lesions from alveolar hyperplasia (AH) to grade 5 tumors shows percent of Glut1 staining defined as weak, intermediate or strong. The number of lesions monitored per grade is indicated. ( b ) Representative Glut1 staining in a KP lung, showing weak expression in the alveolar compartment and strong staining in the bronchiolar epithelium. Scale bar: 20 μm. ( c ) Mouse models and viral vectors used. ( d ) Validation of Glut1 or Glut3 deletion by (upper panels) real-time PCR from purified CD45 - tumor fractions (KP & KPG1: n = 19, 32, respectively; KP & KPG3: n = 14, 13, respectively; KP & KPG1G3: n = 8, 10, respectively) or by (lower panels) Glut1 or Glut3 staining with IHC. ns: not significant by Mann-Whitney test; *: p < 0.05 by Mann-Whitney test; ****: p < 0.0001 by Mann-Whitney test. Scale bars: 200 μm (KP, KPG1 and KPG3 rows). Scale bars: 100 μm (KPG1G3 row). Figure 1—figure supplement 2—source data 1. Source files for Glut1 protein expression analysis by KP tumor grade.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Staining, Immunohistochemistry, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Purification, MANN-WHITNEY

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) (left) Tumor-derived single cell preparations were placed in a Seahorse XF analyzer and subjected to longitudinal extracellular acidification rate (ECAR) measurements. Glucose or 2-deoxyglucose (2-DG) were added where indicated. Data are means ± s.e.m. of 3 KP and 3 KPG1 tumor-derived single cells each analyzed in ten or five technical replicates. The data from the outlier KPG1 are shown separately (orange) from the 2 other KPG1 tumors (red). (right) Real-time PCR and western blot analyses of Glut1 and Glut3 from the samples analyzed by Seahorse. ( b ) Bile acid metabolism genes from Hallmark collection (mSigDB) induced in KPG1 compared to KP from SPC-Cre, CC10-Cre and PGK-Cre tumors. Expression above the median is shown in red, below in green. Genes in red are PPARα targets . ( c ) Gene Set Enrichment Analysis (GSEA) of PPARα targets on genes ranked based on t-statistics when comparing KPG1 and KP tumors in SPC-Cre, CC10-Cre and PGK-Cre. p-values were obtained by normalized enrichment score (NES) of 10 5 random permutations. ( d ) Model illustrating two possible alternatives to Glut1 deficiency for tumor growth. Figure 3—source data 1. Source files for Seahorse analysis of KP and KPG1 tumors.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

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

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Real time PCR for Slc2a3 on KP (n = 19), KPG1 (n = 32), KL (n = 12) and KLG1 (n = 11) tumors. ns: not significant by Mann-Whitney test; ****: p < 0.0001 by Mann-Whitney test. ( b ) Representative examples of Glut3 staining by immunohistochemistry (IHC) from KP, KPG1, KL and KLG1 tumor-bearing lungs. (left panels) Scale bars: 1 mm. (right panels) Scale bars: 100 μm. ( c ) Percent of KP tumors showing Glut3 positive staining in small and big lesions assessed by IHC. ( d ) (left panels) Representative example of H&E, Glut1 or Glut3 staining by IHC from serial sections of a KP tumor shows co-localization. Arrows indicate localization of strong Glut1 and Glut3 expression. Scale bars: 500 μm. (right panel) Percent of Glut3 positive KP tumors showing Glut1 and Glut3 expression correlation, no correlation or anti-correlation assessed by IHC.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Real-time Polymerase Chain Reaction, MANN-WHITNEY, Staining, Immunohistochemistry, Expressing

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Representative examples of H&E, Glut1 or Glut3 staining by IHC from serial sections of a KP tumor showing Glut1-3 co-localization. Scale bars: 200 μm. ( b ) Histogram with mean ± s.d. shows the average number of KP and KPG1G3 tumors per mouse (n = 4 and 6 mice). *: p < 0.05 by Mann-Whitney test. ( c ) Graph with mean ± s.e.m. shows the fold changes of KP and KPG1G3 tumor volumes (n = 8 and 9 tumors) monitored during 43 days by μCT, starting at 15 weeks and 2 days post-tumor initiation with tumor volumes set to 1. ns: not significant by Mann-Whitney test; *: p < 0.05 by Mann-Whitney test. ( d ) Dot plot with mean ± s.d. shows tumor areas in KP and KPG1G3 mice (n = 115 and 101 tumors) calculated from lung sections. **: p < 0.01 by Mann-Whitney test. ( e ) Dot plot with mean ± s.d. shows KP and KPG1G3 tumor weights (n = 8 and 9 tumors, respectively) at sacrifice. **: p < 0.01 by Mann-Whitney test. ( f ) Percent of KP (n = 150) and KPG1G3 (n = 170) lesions classified by tumor grades, either detailed from alveolar hyperplasia (AH) to grade 5 or discriminated between alveolar hyperplasia and adenomas, and adenocarcinomas. Alveolar hyperplasia and adenomas include the AH and the tumor grades 1, 2, and 3. Adenocarcinomas contain the tumor grades 4 and 5. *: p < 0.05; **: p < 0.01. Fisher test was applied when comparing AH, grade 1, grade 2, grade 3, grade 4, and grade 5. Chi-square for trend was applied when comparing alveolar hyperplasia and adenomas, and adenocarcinomas. ( g ) Kaplan-Meier survival analysis of KP (n = 8) and KPG1G3 (n = 6) mice. *: p < 0.05 by Log-rank test. ( h ) Representative (upper panels) positron emission tomography (PET) scan and (lower panels) μCT scan images illustrating the 18 F-FDG absorption of KP, KPG1, KPG3 and KPG1G3 tumors. ( i ) Dot plot with mean ± s.d. displays maximum standardized uptake value (SUV max ) of KP (n = 17), KPG1 (n = 24), KPG3 (n = 32) and KPG1G3 (n = 38) lesions. ns: not significant by Mann-Whitney test; ****: p < 0.0001 by Mann-Whitney test. ( j ) Model illustrating the dependency on glucose transporters for KP and KL tumor progression. Figure 4—source data 1. Source files for tumor growth, grades and survival of KP and KPG1G3 mice.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Staining, MANN-WHITNEY, Positron Emission Tomography

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Dot plots with mean ± s.d. show the relative glucose transporter mRNA expression in CD45 negative fraction from KP and KPG1G3 tumors ( Slc2a1 and Slc2a3 : n = 8 and 10 tumors, respectively; Slc2a2 , Slc2a4 , Slc2a5 , Slc2a6 , Slc2a7 , Slc2a8 , Slc2a9 , Slc2a10 , Slc2a12 , Slc2a13 and Slc50a1 : n = 7 and 3 tumors, respectively). ns: not significant by Mann-Whitney test; ****: p < 0.0001 by Mann-Whitney test

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet: References of the siRNAs used for in vitro experiments.

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: In Vitro

Journal: eLife

Article Title: Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

doi: 10.7554/eLife.53618

Figure Lengend Snippet:

Article Snippet: si SLC2A1 + si SLC2A3 , si SLC2A1 #1 , Human , ThermoFisher Scientific , s12926 Cat# 4390824.

Techniques: Transfection, Construct, Recombinant, Plasmid Preparation, Produced, Clone Assay, Sequencing, FACS, Software

Journal: Cell metabolism

Article Title: The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.

doi: 10.1016/j.cmet.2022.02.015

Figure Lengend Snippet: Figure 1. GLUT3 is required for the effector function of Th17 cells (A) Immunoblot analysis of murine GLUT3, ACLY, IRF4, NFATc1, and GAPDH expression. (B) Analysis of Slc2a3 (GLUT3) gene expression in naive CD4+ T cells and T helper (Th) cell subsets by qRT-PCR; mean ± SEM of 5–6 mice. (C and D) Glycolytic proton efflux rate (glycoPER) analyses of WT and GLUT3-deficient Th1 (C) and Th17 (D) cells using a Seahorse extracellular flux analyzer; mean ± SEM of 5 mice. (E) Proliferation analysis of WT and GLUT3-deficient Th1 and Th17 cells.

Article Snippet: After an 1 h incubation at RT with mouse-anti-Tom20 (Abcam, clone EPR15581-39) and rabbit-anti-human GLUT3 (BioRad), the samples were washed and stained for 1 h with secondary donkey anti-rabbit IgG (H+L) Alexa Fluor 488 and donkey anti-mouse IgG (H+L) Alexa Fluor 647 antibodies (both Invitrogen).

Techniques: Western Blot, Expressing, Gene Expression, Quantitative RT-PCR

Journal: Cell metabolism

Article Title: The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.

doi: 10.1016/j.cmet.2022.02.015

Figure Lengend Snippet: Figure 2. Ablation of GLUT3 in T cells prevents autoimmunity (A–D) Slc2a3fl/flCd4Cre mice are protected from experimental autoimmune encephalomyelitis (EAE). (A) Clinical EAE scores of WT and Slc2a3fl/flCd4Cre mice after immunization with MOG35-55 peptide emulsified in CFA; mean ± SEM of 9 mice per cohort.

Article Snippet: After an 1 h incubation at RT with mouse-anti-Tom20 (Abcam, clone EPR15581-39) and rabbit-anti-human GLUT3 (BioRad), the samples were washed and stained for 1 h with secondary donkey anti-rabbit IgG (H+L) Alexa Fluor 488 and donkey anti-mouse IgG (H+L) Alexa Fluor 647 antibodies (both Invitrogen).

Techniques:

Journal: Cell metabolism

Article Title: The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.

doi: 10.1016/j.cmet.2022.02.015

Figure Lengend Snippet: Figure 3. GLUT3 controls a complex metabolic-transcriptional network in Th17 cells (A) Principal component (PC) analysis of WT and GLUT3-deficient (Slc2a3fl/flCd4Cre) Th1 and Th17 cell RNA-seq data; n = 3 biological replicates per T cell subset and genotype. (B and C) MA plots of differentially expressed genes (DEGs) in WT versus GLUT3-deficient Th1 (B) and Th17 cells (C); genes significantly (p adjusted < 0.01) upregulated and downregulated are depicted in red and blue, respectively. (D) Venn diagram analyses of >4-fold DEGs (p adjusted < 0.01) of GLUT3-deficient Th1 and Th17 cells. (E) Gene set enrichment analysis (GSEA) of WT versus GLUT3-deficient Th17 cells. (F) Network clustering of significantly (p < 0.005) enriched gene expression signatures to identify dysregulated physiological processes in GLUT3-deficient Th17 cells. Downegulated and upregulated gene sets in GLUT3-deficient Th17 cells compared with WT are shown in blue and red, respectively. (G) Heatmap analysis of selected genes in GLUT3-deficient and WT Th1 and Th17 cells. (H) GSEAs of WT versus GLUT3-deficient Th17 cells highlight impaired mitochondrial gene expression and function.

Article Snippet: After an 1 h incubation at RT with mouse-anti-Tom20 (Abcam, clone EPR15581-39) and rabbit-anti-human GLUT3 (BioRad), the samples were washed and stained for 1 h with secondary donkey anti-rabbit IgG (H+L) Alexa Fluor 488 and donkey anti-mouse IgG (H+L) Alexa Fluor 647 antibodies (both Invitrogen).

Techniques: RNA Sequencing, Gene Expression