Journal: iScience

Article Title: Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease

doi: 10.1016/j.isci.2021.103702

Figure Lengend Snippet: Neuronal and glial expression of 128QHtt results in transposable element derepression (A) Semiquantitative RT-PCR analysis to assess the induction levels of the HD transgenic construct by elav-Gal4. cDNA was prepared from total RNA purified from HD ( elav-G4>128QHtt ) and control ( elav-G4/+ ) head tissues. The constitutive gapdh was examined as an endogenous control. (B) qRT-PCR analysis of transposable element expression in larval and adult brains of flies expressing 128QHtt in neurons ( elav-G4>128QHtt ); adult brains were analyzed at both young (0–2 days) and aged (10–12 days) time points; transcript levels were normalized to rp49 and displayed as fold change relative to flies carrying the elav-Gal4 driver with no 128QHtt transgene ( elav-G4/+ ). (C) Western blot assay of gypsy envelope protein (ENV) expression in HD larval and adult brains. GIOTTO protein was used as a loading control. Result was expressed as means for at least three independent biological replicates (∗p < 0.05; one-sample t test). (D) qRT-PCR analysis of TE expression in larval brains and adult heads isolated from 0- to 2-day-old and 10- to 12-day-old flies expressing 128QHtt with the pan-glial repo-Gal4 driver ( repo-Gal4>128QHtt ). Transcript levels were normalized to gapdh and displayed as fold change relative to flies carrying the repo-Gal4 driver with no128QHtt transgene ( repo-G4/+ ). (B and D) Bar graph represents the mean ± SEM from at least three independent experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; unpaired t tests). Red dots indicate individual data points. The black horizontal line indicates the fold change control value, set to 1. See also .

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with the following antibodies diluted in TBST: mouse anti-ENV 8E7 (1:500 , kindly provided by J. Gall), rabbit anti-GIOTTO (1:10,000 ( )), mouse anti-elav (1:500, 9F8A9 DSHB) and mouse anti-repo (1:500, 8D12 DSHB).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Construct, Purification, Quantitative RT-PCR, Western Blot, Isolation

Journal: iScience

Article Title: Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease

doi: 10.1016/j.isci.2021.103702

Figure Lengend Snippet:

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with the following antibodies diluted in TBST: mouse anti-ENV 8E7 (1:500 , kindly provided by J. Gall), rabbit anti-GIOTTO (1:10,000 ( )), mouse anti-elav (1:500, 9F8A9 DSHB) and mouse anti-repo (1:500, 8D12 DSHB).

Techniques: Recombinant, Multiplex Assay, Gel Extraction, Picogreen Assay, SYBR Green Assay, Over Expression, Marker, CNV Assay, Software

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