sirna against slc2a3 (si-slc2a3, sequence c c u g a g a a g a u c a u a a a g g a a t t)  (Thermo Fisher)

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: 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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

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:

Article Snippet: Transfected construct ( Homo sapiens ) , si SLC2A1 #1 , ThermoFisher Scientific , s12926 Cat# 4390824 , Transfected construct.

Techniques: Transfection, Construct, Recombinant, Plasmid Preparation, Produced, Clone Assay, Sequencing, FACS, 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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 SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 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:

Article Snippet: , si SLC2A3 #2 , Human , ThermoFisher Scientific , s12932 Cat# 4390824.

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

Journal: eLife

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

doi: 10.7554/eLife.53618

Figure Lengend Snippet: ( a ) Mouse models and viral vector used for in vivo PPARαΔ13 induction in tumor cells. ( b ) Western blot from transfected 293T cells’ extracts validating the doxycycline-mediated PPARαΔ13 induction in presence of a co-transfected rtTA construct, indicated by the asterisk. ( c ) Graph with mean ± s.e.m. shows the fold changes of KP, KP-PPARαΔ13, KPG1 and KPG1-PPARαΔ13 (n = 11, 16, 11 and 8, respectively) tumor volumes monitored during 23 days by μCT, before and during doxycycline diet, starting at 22 weeks and 4 days post-tumor initiation with tumor volumes set to 1. ns: not significant by Mann-Whitney test. ( d ) Hallmark and KEGG pathways regulated (induced in red and repressed in blue) by PPARαΔ13 versus control in KP (top panels) and KPG1 (bottom panels) tumors. Bar length represents the normalized enrichment score (NES) obtained by GSEA. Genesets with significant enrichment (p-value < 0.05) are shown. Figure 3—figure supplement 3—source data 1. Source files for tumor growth of KP and KPG1 mice, with or without PPARαΔ13 induction.

Article Snippet: Transfected construct ( Homo sapiens ) , si SLC2A3 #1 , ThermoFisher Scientific , s12931 Cat# 4390824 , Transfected construct.

Techniques: Plasmid Preparation, In Vivo, Western Blot, Transfection, Construct, MANN-WHITNEY, Control

Journal: eLife

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

doi: 10.7554/eLife.53618

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapiens ) , si SLC2A3 #1 , ThermoFisher Scientific , s12931 Cat# 4390824 , Transfected construct.

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