Journal: JCI Insight

Article Title: A muscle-specific UBE2O/AMPK α 2 axis promotes insulin resistance and metabolic syndrome in obesity

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: (A) An RNA interference (RNAi) screen identifies UBE2O as a potent regulator of glucose uptake in myotubes. Primary normal human skeletal myotubes (HSkMs) and C2C12 mouse myotubes expressing a nontargeting control siRNA pool (Cont. siRNAs) or a synthetic siRNA library targeting 30 human and mouse E2s, respectively, were subjected to a screen for insulin-stimulated 2-deoxyglucose uptake. 2-DG6P, 2-deoxyglucose-6-phosphate. n = 3. (B) Total RNAs from quadriceps skeletal muscles of mice fed a normal chow or an HFD for 28 weeks were subjected to RT-qPCR. n = 4. (C and D) Lysates from quadriceps skeletal muscles of 9-month-old Ube2o+/+ mice fed normal chow or an HFD for 28 weeks (C) and 25-week-old type 2 diabetic db/db and lean (db/+) mice (D) were subjected to immunoblotting. Error bars represent ±SEM. P value was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, control siRNAs HSkMs vs. E2 siRNAs HSkMs; #P < 0.05, ##P < 0.01, ###P < 0.001, control siRNAs C2C12 vs. E2 siRNAs C2C12; †P < 0.05, †††P < 0.001, chow vs. HFD. Uncropped gels and blots are available in the supplemental material.

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: JCI Insight

Article Title: A muscle-specific UBE2O/AMPK α 2 axis promotes insulin resistance and metabolic syndrome in obesity

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: (A) Body weight increase of Ube2o+/+ and Ube2o–/– mice fed an HFD for 21 weeks. Ube2o+/+ n = 7, Ube2o–/– n = 9. (B) Food intake (grams per day) in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 6, Ube2o–/– n = 8. (C) Fat mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 5, Ube2o–/– n = 9. (D) Adipose depot weight of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; BAT, brown adipose tissue. Ube2o+/+ n = 6, Ube2o–/– n = 9. (E) H&E-stained sections of SAT in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Scale bars: 75 μm. (F) Plasma levels of total cholesterol (TC) (mg/dL), free fatty acid (FFA; mEq/L), HDL (mg/dL), LDL (mg/dL) in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. n = 6. (G) Liver weight of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 6, Ube2o–/– n = 9. (H) Hepatic triglyceride (TG) level per gram of liver in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. n = 6. (I) H&E-stained sections of liver in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Scale bars: 75 μm. (J) Oxygen consumption (VO2) per kg lean mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (K) Respiratory exchange ratio (RER; VCO2/VO2) of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (L) Energy expenditure during day and night per kg of lean mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (M) Home cage locomotor activity during both the light and dark phases of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined by using an automated combined indirect calorimetry system. Ube2o+/+ n = 5, Ube2o–/– n = 8. (N) Rectal temperature of Ube2o+/+ and Ube2o–/– mice on an HFD for 12 weeks. Ube2o+/+ n = 8, Ube2o–/– n = 9. Error bars represent ±SEM. P value was determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Staining, Activity Assay

Journal: JCI Insight

Article Title: A muscle-specific UBE2O/AMPK α 2 axis promotes insulin resistance and metabolic syndrome in obesity

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: (A) Blood glucose was measured Ube2o+/+ and Ube2o–/– mice on an HFD for 21 weeks. n = 6. (B and C) Glucose (GTT) (B) and insulin (ITT) (C) tolerance tests in Ube2o+/+ and Ube2o–/– mice on HFD for 21~22 weeks. Insets indicate AUC (B) and area above the curve (AAC) (C). Insulinemia at 15 minutes after the injection of glucose during a GTT test is shown in the right panel (B). Ube2o+/+ n = 6, Ube2o–/– n = 9. (D–F) Rates of basal and clamp endogenous glucose production (D), glucose infusion (E), and glucose disposal rate (F) during a hyperinsulinemic-euglycemic clamp study in Ube2o+/+ and Ube2o–/– mice. n = 6. (G) Insulin-stimulated 14C-2-deoxyglucose uptake was assessed in visceral WAT and gastrocnemius muscle of Ube2o+/+ and Ube2o–/– mice during the final 45 minutes of the hyperinsulinemic-euglycemic clamps. n = 7. (H) Immunoblots (left) and statistical data (right) showing insulin-induced (200 nM) tyrosine phosphorylation of IRS1 immunoprecipitates and S473 phosphorylation of AKT protein and their total protein levels in the extensor digitorum longus muscle from Ube2o+/+ and Ube2o–/– mice on an HFD. n = 3. Error bars represent ± =SEM. P value was determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Injection, Western Blot

Journal: JCI Insight

Article Title: A muscle-specific UBE2O/AMPK α 2 axis promotes insulin resistance and metabolic syndrome in obesity

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: (A) UBE2O (top) or AMPKα2 (bottom) immunoprecipitates of lysates from skeletal muscle of control (Ube2ofl/fl) and Ube2oΔMus mice were subjected to immunoblotting for AMPKα2 or UBE2O. (B) Lysates from C2C12 myotubes transfected with the indicated plasmids were subjected to metal affinity purification for His-tagged ubiquitin (His-Ub), then immunoblotting for ubiquitinated AMPKα2. Ni-NTA, Ni2+-nitrilotriacetic acid. (C) Recombinant AMPKα2 proteins were subjected to in vitro ubiquitination assay in the presence of in vitro translated WT or C1037S (CS) mutant UBE2O. (D) Lysates from C2C12 myotubes expressing WT or CS mutant UBE2O treated with MG132 (10 μM) for 6 hours were subjected to immunoblotting. (E) Lysates from C2C12 myotubes expressing WT or CS mutant UBE2O treated with cycloheximide (CHX) for the indicated times were subjected to immunoblotting (top). AMPKα2 or AMPKα1 protein levels were quantified by normalizing to the intensity of the HSP90 band (bottom). (F) Lysates from skeletal muscle of 12-week-old Ube2o+/+, Ube2o–/–, Ube2ofl/fl, and Ube2oΔMus mice were subjected to immunoblotting for the indicated proteins. (G) Lysates from C2C12 myotubes expressing UBE2O shRNA together with exogenous WT or CS mutant UBE2O from a Ube2o ORF transcript lacking the 3′ UTR sequence targeted by shRNA (shUbe2o-3′UTR) were subjected to immunoblotting for the indicated proteins. (H) Insulin-stimulated glucose uptake rate of C2C12 myotubes from G. 2-DG6P, 2-deoxyglucose-6-phosphate. n = 4. (I) Lysates from C2C12 myotubes expressing Ube2o shRNA together with Prkaa2 shRNA were subjected to immunoblotting for the indicated proteins. (J) Insulin-stimulated glucose uptake rate of C2C12 myotubes from I. n = 3. Error bars represent ±SEM. P value was determined by ANOVA (***P < 0.001, ###P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Western Blot, Transfection, Affinity Purification, Recombinant, In Vitro, Ubiquitin Assay, Mutagenesis, Expressing, shRNA, Sequencing

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Viability curves of A2780 cell lines transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2). Each experimental group was exposed to six different CDDP concentrations for 48h. Data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. (B) Viability of A2780 cell lines transfected with pCMV6 (R-Ø) and with the overexpression vectors (R-ITF2). (C) Relative expression levels of ITF2 measured by quantitative RT-PCR, represented in Log10 scale; In each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. (D) β-catenin transcriptional activity was measured in A2780 cells after ITF2 overexpression and treatment with LiCl (10mM) for 24 hours, transfecting with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (=1). Values represent the mean of three independent experiments measured by triplicate ± SD. (E) Expression analysis of the downstream gene DKK1 regulated by ITF2 in A2780 cell line transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2) for 24 and 72 hours. Representative images of DKK1 and GAPDH measured by RT-PCR. (F), Expression levels of DKK1 measured by qRT-PCR. Each assay was performed at least three times to confirm the results. *** p < 0.001; * p < 0.05 (Students T-test).

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Transfection, Over Expression, Plasmid Preparation, Concentration Assay, Expressing, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Correlation between ITF2 expression and HOXD9 in tumor and non-tumor samples from the complete cohort of 25 NSCLC patients. Pearson coefficient was used for linear correlation of the quantitative variables. (B-C) Effect of ITF2 overexpression on HOXD9 levels (B) Validation of the transfection efficiency of ITF2 at mRNA levels. Relative expression levels of ITF2 measured by qRT-PCR, in the cell line H23R, at 24 and 72 hours after transfectionrepresented in Log10 the 2-ΔΔCt. (C) Relative expression levels of HOXD9 measured by quantitative RT-PCR after ITF2 overexpression. For both (B) and (C) the resistant cell line transfected with pCMV6 plasmid was used as a calibrator (R-Ø). H23R cells were also transfected with ITF2 cDNA (R-ITF2). Each bar represents the combined relative expression of two independent experiments measured in triplicate. *** p < 0.001; ** p < 0.01 (Students T-test). (D) Correlation between ITF2 and HOXD9 expression levels with the overall survival analyzed in NSCLC patients selected form the RNA-seq analysis. Data represents the quantitative expression levels of the two genes measured by qRT-PCR and represented as 2-ΔΔCt for ITF2 (referred to the NLM) and 2-ΔCt for HOXD9 . (E) Survival analysis in NSCLC samples according to the mean of HOXD9 . LogRank, Breslow and Tarone-Ware test were used for comparisons and p < 0.05 was considered as a significant change in OS. (F-H) Survival analysis in 1,926 patients from the Kaplan Meier online tool (F) and the TCGA data sets of lung Adenocarcinoma (G, High=23; No Change=203) and SCC (H, High=28; No Change=441) for HOXD9 .

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Expressing, Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation, RNA Sequencing Assay

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 1. Physical Interactions Between Hippo Signaling Complex, PCBP1 and PCBP2 in 601

Article Snippet: 150 151 PCBP2 mutations-Full-length PCBP2 (isoform a) was constructed from the PCBP2 plasmid 152 (isoform b, Origene)(10).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 3: Degradation of Mst1 with PCBP2 Depletion in MCF10A Cells 635

Article Snippet: 150 151 PCBP2 mutations-Full-length PCBP2 (isoform a) was constructed from the PCBP2 plasmid 152 (isoform b, Origene)(10).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 4: Loss of Lats and YAP Phosphorylation, Increased TAZ Levels with PCBP2 652

Article Snippet: 150 151 PCBP2 mutations-Full-length PCBP2 (isoform a) was constructed from the PCBP2 plasmid 152 (isoform b, Origene)(10).

Techniques: Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 5. TAZ stabilization with PCBP2 depletion. A. Equalization of TAZ levels in control 674

Article Snippet: 150 151 PCBP2 mutations-Full-length PCBP2 (isoform a) was constructed from the PCBP2 plasmid 152 (isoform b, Origene)(10).

Techniques: Control

Journal: Molecular and Cellular Biology

Article Title: Poly(rC)-Binding Protein 2 Regulates Hippo Signaling To Control Growth in Breast Epithelial Cells

doi: 10.1128/mcb.00104-16

Figure Lengend Snippet: Figure 7: Requirement of YAP and TAZ for PCBP2-mediated Changes in Transcription. 701

Article Snippet: 150 151 PCBP2 mutations-Full-length PCBP2 (isoform a) was constructed from the PCBP2 plasmid 152 (isoform b, Origene)(10).

Techniques:

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 1. UBE2O is upregulated in obese subjects with type 2 diabetes. (A) An RNA interference (RNAi) screen identifies UBE2O as a potent regulator of glucose uptake in myotubes. Primary normal human skeletal myotubes (HSkMs) and C2C12 mouse myotubes expressing a nontargeting control siRNA pool (Cont. siRNAs) or a synthetic siRNA library targeting 30 human and mouse E2s, respectively, were subjected to a screen for insulin-stimulated 2-deoxyglucose uptake. 2-DG6P, 2-deoxyglucose-6-phosphate. n = 3. (B) Total RNAs from quadriceps skeletal muscles of mice fed a normal chow or an HFD for 28 weeks were subjected to RT-qPCR. n = 4. (C and D) Lysates from quadriceps skeletal muscles of 9-month-old Ube2o+/+ mice fed normal chow or an HFD for 28 weeks (C) and 25-week- old type 2 diabetic db/db and lean (db/+) mice (D) were subjected to immunoblotting. Error bars represent ±SEM. P value was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, control siRNAs HSkMs vs. E2 siRNAs HSkMs; #P < 0.05, ##P < 0.01, ###P < 0.001, control siRNAs C2C12 vs. E2 siRNAs C2C12; †P < 0.05, †††P < 0.001, chow vs. HFD. Uncropped gels and blots are available in the supplemental material.

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Expressing, Control, Muscles, Quantitative RT-PCR, Western Blot

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 2. Ube2o ablation protects mice against diet-induced obesity and metabolic syndrome. (A) Body weight increase of Ube2o+/+ and Ube2o–/– mice fed an HFD for 21 weeks. Ube2o+/+ n = 7, Ube2o–/– n = 9. (B) Food intake (grams per day) in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 6, Ube2o–/– n = 8. (C) Fat mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 5, Ube2o–/– n = 9. (D) Adipose depot weight of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; BAT, brown adipose tissue. Ube2o+/+ n = 6, Ube2o–/– n = 9. (E) H&E-stained sections of SAT in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Scale bars: 75 μm. (F) Plasma levels of total cholesterol (TC) (mg/dL), free fatty acid (FFA; mEq/L), HDL (mg/dL), LDL (mg/dL) in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. n = 6. (G) Liver weight of Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Ube2o+/+ n = 6, Ube2o–/– n = 9. (H) Hepatic triglyceride (TG) level per gram of liver in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. n = 6. (I) H&E-stained sections of liver in Ube2o+/+ and Ube2o–/– mice on an HFD for 28 weeks. Scale bars: 75 μm. (J) Oxygen consumption (VO2) per kg lean mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (K) Respiratory exchange ratio (RER; VCO2/VO2) of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (L) Energy expenditure during day and night per kg of lean mass of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined in metabolic chambers. Ube2o+/+ n = 6, Ube2o–/– n = 9. (M) Home cage locomotor activity during both the light and dark phases of Ube2o+/+ and Ube2o–/– mice on an HFD for 20 weeks was determined by using an automated combined indirect calorimetry system. Ube2o+/+ n = 5, Ube2o–/– n = 8. (N) Rectal temperature of Ube2o+/+ and Ube2o–/– mice on an HFD for 12 weeks. Ube2o+/+ n = 8, Ube2o–/– n = 9. Error bars represent ±SEM. P value was determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Staining, Clinical Proteomics, Activity Assay

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 3. Ube2o–/– mice show improved insulin sensitivity in diet-induced type 2 diabetes mouse model. (A) Blood glucose was measured Ube2o+/+ and Ube2o–/– mice on an HFD for 21 weeks. n = 6. (B and C) Glucose (GTT) (B) and insulin (ITT) (C) tolerance tests in Ube2o+/+ and Ube2o–/– mice on HFD for 21~22 weeks. Insets indicate AUC (B) and area above the curve (AAC) (C). Insulinemia at 15 minutes after the injection of glucose during a GTT test is shown in the right panel (B). Ube2o+/+ n = 6, Ube2o–/– n = 9. (D–F) Rates of basal and clamp endogenous glucose production (D), glucose infusion (E), and glucose disposal rate (F) during a hyperinsulinemic-euglycemic clamp study in Ube2o+/+ and Ube2o–/– mice. n = 6. (G) Insulin-stimulated 14C-2-deoxyglucose uptake was assessed in visceral WAT and gastrocnemius muscle of Ube2o+/+ and Ube2o–/– mice during the final 45 minutes of the hyperinsulinemic-euglycemic clamps. n = 7. (H) Immunoblots (left) and statistical data (right) showing insulin-induced (200 nM) tyrosine phosphorylation of IRS1 immunoprecipitates and S473 phosphorylation of AKT protein and their total protein levels in the extensor digitorum longus muscle from Ube2o+/+ and Ube2o–/– mice on an HFD. n = 3. Error bars represent ± =SEM. P value was determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Injection, Western Blot, Phospho-proteomics

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 4. Muscle-specific ablation of Ube2o blocks diet-induced insulin resistance and metabolic disorders. (A) Body weights of control (Ube2ofl/fl) and Ube2oΔMus mice fed an HFD for 20 weeks. Ube2ofl/fl n = 12, Ube2oΔMus n = 8. (B) Food intake (grams per day) in control and Ube2oΔMus mice on an HFD for 16 weeks. n = 7. (C) Fat mass of control and Ube2oΔMus mice on HFD for 20 weeks. n = 7. (D) H&E-stained sections of subcutaneous adi- pose tissue in control and Ube2oΔMus mice on an HFD for 20 weeks. Scale bars: 75 μm. (E) Plasma levels of TC (mg/dL), FFA (mEq/L), HDL (mg/dL), and LDL (mg/dL) in control and Ube2oΔMus mice on an HFD for 20 weeks. n = 7. (F) Photographs and H&E-stained sections of liver in control and Ube2oΔMus mice on an HFD for 20 weeks. Scale bars: 75 μm. (G) Liver weight of control and Ube2oΔMus mice on an HFD for 20 weeks. Ube2ofl/fl n = 6, Ube2oΔMus n = 4. (H) Hepatic TG level per gram of liver in control and Ube2oΔMus mice on an HFD for 20 weeks. Ube2ofl/fl n = 6, Ube2oΔMus n = 4. (I) Total RNAs from WAT, skeletal muscle, and BAT of control and Ube2oΔMus mice on an HFD for 20 weeks were subjected to RT-qPCR. n = 6. (J) VO2 per kg of lean mass in control and Ube2oΔMus mice was determined in metabolic chambers. n = 7. (K) RER (VCO2/VO2) of control and Ube2oΔMus mice was deter- mined in metabolic chambers. n = 7. (L) Energy expenditure during day and night per kg of lean mass of control and Ube2oΔMus mice was determined in metabolic chambers. n = 7. (M) Home cage locomotor activity during both the light and dark phases of control and Ube2oΔMus mice was determined

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Control, Staining, Clinical Proteomics, Quantitative RT-PCR, Activity Assay

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 5. Adipose tissue–specific Ube2o-knockout mice do not have improved fat metabolism on an HFD. (A) Body weights of control (Ube2ofl/fl) and Ube2oΔAdip mice fed an HFD for 20 weeks. n = 10. (B) Adipose depot weight of control and Ube2oΔAdip mice on an HFD for 20 weeks. SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; BAT, brown adipose tissue. Ube2ofl/fl n = 8, Ube2oΔAdip n = 10. (C) H&E-stained sections of SAT in control and Ube2oΔAdip mice on an HFD for 20 weeks. Scale bars: 75 μm. (D) Plasma levels of TC (mg/dL) in control and Ube2oΔAdip mice on an HFD for 20 weeks. Ube2ofl/fl n = 8, Ube2oΔAdip n = 10. (E) Liver weight of control and Ube2oΔAdip mice on an HFD for 20 weeks. Ube2ofl/fl n = 8, Ube2oΔAdip n = 10. (F) Hepatic TG level per gram of liver in control and Ube2oΔAdip mice on an HFD for 20 weeks. Ube2ofl/fl n = 8, Ube2oΔAdip n = 9. (G) H&E-stained sections of liver in control and Ube2oΔAdip mice on an HFD for 20 weeks. Scale bars: 75 μm. GTTs (H) and ITTs (I) in control and Ube2oΔAdip mice on an HFD for 16 weeks. Insets indicate AUC (H) and AAC (I). n = 9. (J) Immuno- blots showing insulin-induced (200 nM) tyrosine phosphorylation of IRS1 immunoprecipitates and S473 phosphorylation of AKT protein and their total protein levels in VAT from control and Ube2oΔAdip mice on an HFD for 20 weeks. Error bars represent ±SEM. P value was determined by Student’s t test.

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Knock-Out, Control, Staining, Clinical Proteomics, Western Blot, Phospho-proteomics

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 6. Regulation of AMPKα2 by skeletal muscle UBE2O. (A) UBE2O (top) or AMPKα2 (bottom) immunoprecip- itates of lysates from skeletal muscle of control (Ube2ofl/fl) and Ube2oΔMus mice were subjected to immunoblotting for AMPKα2 or UBE2O. (B) Lysates from C2C12 myotubes transfected with the indicated plasmids were subjected to metal affinity purification for His-tagged ubiquitin (His-Ub), then immunoblotting for ubiquitinated AMPKα2. Ni-NTA, Ni2+-nitrilotriacetic acid. (C) Recombinant AMPKα2 proteins were subjected to in vitro ubiquitination assay in the presence of in vitro translated WT or C1037S (CS) mutant UBE2O. (D) Lysates from C2C12 myotubes express- ing WT or CS mutant UBE2O treated with MG132 (10 μM) for 6 hours were subjected to immunoblotting. (E) Lysates from C2C12 myotubes expressing WT or CS mutant UBE2O treated with cycloheximide (CHX) for the indicated times were subjected to immunoblotting (top). AMPKα2 or AMPKα1 protein levels were quantified by normalizing to the intensity of the HSP90 band (bottom). (F) Lysates from skeletal muscle of 12-week-old Ube2o+/+, Ube2o–/–, Ube2ofl/fl, and Ube2oΔMus mice were subjected to immunoblotting for the indicated proteins. (G) Lysates from C2C12 myotubes expressing UBE2O shRNA together with exogenous WT or CS mutant UBE2O from a Ube2o ORF transcript lacking the 3′ UTR sequence targeted by shRNA (shUbe2o-3′UTR) were subjected to immunoblotting for the indicated proteins. (H) Insulin-stimulated glucose uptake rate of C2C12 myotubes from G. 2-DG6P, 2-deoxyglu- cose-6-phosphate. n = 4. (I) Lysates from C2C12 myotubes expressing Ube2o shRNA together with Prkaa2 shRNA were subjected to immunoblotting for the indicated proteins. (J) Insulin-stimulated glucose uptake rate of C2C12 myotubes from I. n = 3. Error bars represent ±SEM. P value was determined by ANOVA (***P < 0.001, ###P < 0.001).

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Control, Western Blot, Transfection, Affinity Purification, Ubiquitin Proteomics, Recombinant, In Vitro, Mutagenesis, Expressing, shRNA, Sequencing

Journal: JCI insight

Article Title: A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

doi: 10.1172/jci.insight.128269

Figure Lengend Snippet: Figure 7. Activation of skeletal muscle AMPKα2 drives the protective effects of UBE2O loss on diabetes and metabolic disorders. (A) Body weights of control (Ube2ofl/fl), Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice fed an HFD for 20 weeks. Ube2ofl/fl n = 13, Ube2oΔMus n = 7, ΔMus;Prkaa2Δ/+ n = 7. (B) Percentage of fat and lean mass of control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 16 weeks was determined by EchoMRI. Ube2ofl/fl n = 6, Ube2oΔMus n = 3, ΔMus;Prkaa2Δ/+ n = 5. (C) H&E-stained sections of SAT in control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks. Scale bars: 75 μm. (D) Plasma levels of TC (mg/dL) in control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks. Ube2ofl/fl n = 5, Ube2oΔMus n = 3, ΔMus;Prkaa2Δ/+ n = 5. (E) Photographs and H&E-stained sections of liver in control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks. Scale bars: 75 μm. (F) Liver weight of control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks. Ube2ofl/fl n = 6, Ube2oΔMus n = 3, ΔMus;Prkaa2Δ/+ n = 6. (G) Hepatic TG level per g of liver of control, Ube2oΔMus and ΔMus;Prkaa2Δ/+ mice on HFD for 20 weeks. Ube2ofl/fl n = 6, Ube2oΔMus n = 3, ΔMus;Prkaa2Δ/+ n = 6. (H) Total RNAs from WAT and BAT of control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks were subjected to RT-qPCR. n = 4. (I) Lysates from skeletal muscle of control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks were subjected to immunoblotting for the indicated proteins. (J) Total RNAs from skeletal muscle of control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 20 weeks were subjected to RT-qPCR. n = 4. GTTs (K) and ITTs (L) in control, Ube2oΔMus, and ΔMus;Prkaa2Δ/+ mice on an HFD for 9 and 10 weeks, respectively. AUC (K) and AAC (L) are shown in the right panels. Ube2ofl/fl n = 5, Ube2oΔMus n = 3, ΔMus;Prkaa2Δ/+ n = 5. Error bars represent ±SEM. P value was determined by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, Ube2ofl/fl vs. Ube2oΔMus; #P < 0.05, ##P < 0.01, ###P < 0.001, Ube2oΔMus vs. ΔMus;Prkaa2Δ/+; †P < 0.05, Ube2ofl/fl vs. ΔMus;Prkaa2Δ/+.

Article Snippet: The mutant construct (C1037S) of Flag-tagged mouse UBE2O (Origene) was generated by site-directed mutagenesis and cloned into pBabe retroviral vector.

Techniques: Activation Assay, Control, Staining, Clinical Proteomics, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: A Novel Role for the Tumor Suppressor Gene ITF2 in Tumorigenesis and Chemotherapy Response

doi: 10.3390/cancers12040786

Figure Lengend Snippet: Clinicopathological and experimental data obtained from patients with NSCLC from La Paz University Hospital.

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, MD, USA) using previously described methodology [ ].

Techniques: