Journal: Cell Communication and Signaling : CCS

Article Title: LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1

doi: 10.1186/s12964-020-00654-2

Figure Lengend Snippet: H19 overexpression in db/db mice ameliorates glucose intolerance and lipid ectopic accumulation. ( a ) The expression level of H19 was decreased in skeletal muscle of db/db mice. ( b ) Ad-H19 injection resulted in H19 overexpression in skeletal muscle of db/db mice. Fasting glucose levels ( c ) and serum GA levels ( d ) in db/db mice after H19 overexpression. ( e ) Left: OGTTs at 2 weeks after viral injection. Right: area under the curve (AUC) data. ( f ) Fasting serum insulin levels of db/db mice after H19 overexpression. Serum TG ( g ) and Skeletal muscle TG ( h ) levels in db/db mice after H19 overexpression. Representative images of Oil Red O staining assays ( i ) and H&E staining assays ( j ) of db/db mouse skeletal muscle after H19 overexpression. n = 6 mice per genotype. Numbers were the mean ± SD. * p < 0.05, ** p < 0.01, n.s, not significant

Article Snippet: The adenoviruses (Ad) expressing H19 and ad-green fluorescent protein (GFP) were constructed by Shanghai Genechem Company.

Techniques: Over Expression, Expressing, Injection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1

doi: 10.1186/s12964-020-00654-2

Figure Lengend Snippet: H19 regulates lipid metabolism in skeletal muscle cells. ( a ) Real-time PCR analysis of H19 expression in muscle cells treated with ad-H19. DAG contents( b ) and TG contents ( c )in muscle cells in the absence or presence of FFA after H19 overexpression. ( d ) Real-time PCR analysis of H19 expression in muscle cells treated with si-H19. DAG contents( e ) and TG contents (F)in muscle cells in the absence or presence of FFA after H19 knockdown. ( g ) Representative images of Nile red staining assays in muscle cells of H19 overexpression or silencing. ( h ) Representative tracings of OCR measured in the H19 overexpression and control muscle cells. Maximal mitochondrial respiration ( i ), basal mitochondrial respiration ( j ), ATP production ( k ) and proton leak ( l ) of ad-H19 and ad-GFP infected muscle cells. (M) Representative tracings of OCR measured in the H19 knockdown and control muscle cells. Maximal mitochondrial respiration ( n ), basal mitochondrial respiration ( o ), ATP production ( p ) and proton leak ( q ) of si-H19 and si-Con transfected muscle cells. Quantification was based on 3 independent transfection/infection experiments. Numbers were the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: The adenoviruses (Ad) expressing H19 and ad-green fluorescent protein (GFP) were constructed by Shanghai Genechem Company.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Knockdown, Staining, Control, Infection, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1

doi: 10.1186/s12964-020-00654-2

Figure Lengend Snippet: The effects of H19 on metabolic signaling pathway genes in skeletal muscles and cells. ( a ) H19 overexpression increased the protein levels of glucose and lipid metabolic genes in skeletal muscles. Representative gel images are presented in the left panel, and quantitative data are in the right panel. ( b ) H19 overexpression in skeletal muscles increased mRNA of glucose and lipid metabolic genes. H19 overexpression ( c ) and H19 knockdown ( d ) in muscle cells influenced mRNA of glucose and lipid metabolic genes. ( e ) H19 overexpression in muscle cells increased mitochondrial electronic chain complex proteins. n = 6 mice per genotype. Quantification was based on 3 independent experiments. Numbers were the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s, not significant

Article Snippet: The adenoviruses (Ad) expressing H19 and ad-green fluorescent protein (GFP) were constructed by Shanghai Genechem Company.

Techniques: Muscles, Over Expression, Knockdown

Journal: Cell Communication and Signaling : CCS

Article Title: LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1

doi: 10.1186/s12964-020-00654-2

Figure Lengend Snippet: Validation of interaction between hnRNPA1 and H19. a Flow diagram of RNA pull-down assay. b Silver staining result of RNA pull-down assay revealed H19 interacted with hnRNPA1 in normal C2C12 cell lines. c Western blotting identified hnRNPA1 bound with H19. d RNA immunoprecipitation (RIP) revealed that hnRNPA1 was bound with H19. e RIP identified that hnRNPA1 was bound with CPT1b, CD36, PGC1a. Quantification was based on 3 independent transfection/infection experiments. Numbers were the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s, not significant

Article Snippet: The adenoviruses (Ad) expressing H19 and ad-green fluorescent protein (GFP) were constructed by Shanghai Genechem Company.

Techniques: Biomarker Discovery, Pull Down Assay, Silver Staining, Western Blot, RNA Immunoprecipitation, Transfection, Infection

Journal: Cell Communication and Signaling : CCS

Article Title: LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1

doi: 10.1186/s12964-020-00654-2

Figure Lengend Snippet: The effects of H19-hnRNPA1 complexes in metabolism. ( a ) Representative tracings of OCR measured in the hnRNPA1 overexpression and control muscle cells in the presence of H19 knockdown. Basal mitochondrial respiration ( b ), maximal mitochondrial respiration ( c ), ATP production ( d ) and proton leak ( e ) of OE-hnRNPA1 and OE-vector transfected muscle cells in the presence of H19 knockdown. ( f ) The protein levels of PGC1a and CPT1b of OE-hnRNPA1 and Vector transfected muscle cells in the presence of H19 knockdown. ( g ) Proposed model of H19-mediated regulation of lipid contents in skeletal muscle. Quantification was based on 3 independent transfection/infection experiments. Numbers were the mean ± SD. *** p < 0.001, * p < 0.05, n.s, not significant

Article Snippet: The adenoviruses (Ad) expressing H19 and ad-green fluorescent protein (GFP) were constructed by Shanghai Genechem Company.

Techniques: Over Expression, Control, Knockdown, Plasmid Preparation, Transfection, Infection

Journal: The American Journal of Pathology

Article Title: Hepatic Knockdown of Splicing Regulator Slu7 Ameliorates Inflammation and Attenuates Liver Injury in Ethanol-Fed Mice

doi: 10.1016/j.ajpath.2018.05.004

Figure Lengend Snippet: Selected Parameters in Slu7 Overexpression Mice after Ethanol Feeding

Article Snippet: Murine vectors expressing Ad–green fluorescent protein (GFP; control), Ad-Slu7 [adenoviral type 5 (dE1/E3); promoter, cytomegalovirus], Ad-shRNA (control), and Ad-shSlu7 [adenoviral type 5 (dE1/E3); promoter, U6] were custom designed and obtained from Vector BioLabs, Inc. (Malvern, PA).

Techniques: Over Expression

Journal: Reproductive Sciences

Article Title: Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids

doi: 10.1177/1933719116638186

Figure Lengend Snippet: List of Antibodies Used for WB and ChIP Assay Analysis.

Article Snippet: Immortalized human uterine fibroid cells (HuLM) were cultured in 60-mm dishes; at 30% to 40% confluence, cells were transduced with Ad- EZH2 (adenovirus expressing EZH2 under CMV5 promoter; Vector Biolabs, Malvern, Pennsylvania) and Ad- Green Fluorescent Protein (GFP) at varying multiplicity of infections (0-100 plaque-forming unit/cell), as we have described previously.

Techniques: Concentration Assay, Negative Control

Journal: Reproductive Sciences

Article Title: Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids

doi: 10.1177/1933719116638186

Figure Lengend Snippet: Inhibition of EZH2 increases the expression of MSH2 in a dose-dependent manner in human fibroid cells. A, Inhibition of EZH2 by 3-deazaneplanocin A (DZNep) decreased levels of H3K27me3 by Western blot analysis. Primary fibroid cells (PFCs) were plated in 60-mm dish. Cells were treated with varying concentrations of EZH2 inhibitor DZNep for 3 days. Dimethyl sulfoxide (DMSO) was used as a vehicle control. B, The protein lysates were prepared from PFCs treated with vehicle or 1 μmol/L DZNep for 3 days. The protein levels of MSH2 were determined by Western blot analysis. In addition, cells were treated with varying concentrations of EZH2 inhibitor DZNep for 3 days. Cells were collected and subjected to RNA extraction and complementary DNA (cDNA) synthesis. Quantitative polymerase chain reaction was performed to measure the expression levels of MSH2 (C), SUZ12 (D), EED (E), and P27 (F). 18S was used as an endogenous control.

Article Snippet: Immortalized human uterine fibroid cells (HuLM) were cultured in 60-mm dishes; at 30% to 40% confluence, cells were transduced with Ad- EZH2 (adenovirus expressing EZH2 under CMV5 promoter; Vector Biolabs, Malvern, Pennsylvania) and Ad- Green Fluorescent Protein (GFP) at varying multiplicity of infections (0-100 plaque-forming unit/cell), as we have described previously.

Techniques: Inhibition, Expressing, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction

Journal: Reproductive Sciences

Article Title: Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids

doi: 10.1177/1933719116638186

Figure Lengend Snippet: Overexpression of EZH2 increases HuLM cell proliferation. HuLM cells were plated in 96-well dish. Cellular confluence was approximately 30%. Equal amounts of virus (20 plaque-forming unit [pfu]/cell) containing GFP or EZH2 gene were added to the medium, respectively. Eight hours later, the viral supernatant was removed and fresh medium was added. After 3 days and 6 days of infection, MTT assay was performed to determine absorbance at 570 nm. *P < .05 compared with the control.

Article Snippet: Immortalized human uterine fibroid cells (HuLM) were cultured in 60-mm dishes; at 30% to 40% confluence, cells were transduced with Ad- EZH2 (adenovirus expressing EZH2 under CMV5 promoter; Vector Biolabs, Malvern, Pennsylvania) and Ad- Green Fluorescent Protein (GFP) at varying multiplicity of infections (0-100 plaque-forming unit/cell), as we have described previously.

Techniques: Over Expression, Infection, MTT Assay

Journal: Reproductive Sciences

Article Title: Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids

doi: 10.1177/1933719116638186

Figure Lengend Snippet: Overexpression of EZH2 using adenoviral vector decreases MSH2 expression in HuLM cells. A, EZH2 was overexpressed in HuLM cells infected with adenovirus containing EZH2 as compared to cells infected with adenovirus containing GFP. Levels of epigenetic mark H3K27me3 were also increased in HuLM cells infected with virus containing EZH2. B, EZH2 was found in both cytoplasm and nucleus; however, higher expression of EZH2 was observed in the nucleus as compared to the cytoplasm. C, MSH2 RNA expression was downregulated in HuLM cells overexpressed with EZH2 as compared to GFP control. D, Cell cycle regulatory gene p27 is downregulated in EZH2-overexpressed cells as compared to GFP control cells. *P < .05 compared with the control. Total cell lysates (E), nucleus (F), and cytoplasm (G) from HuLM cells infected with Ad-EZH2 or Ad-GFP were analyzed by Western blot using Anti-MSH2 antibody. Western blot with anti–β-actin antibody was used as the loading control. MSH2 protein bands were quantified and normalized to β-actin, and relative values were used to generate data graphs (right panels).

Article Snippet: Immortalized human uterine fibroid cells (HuLM) were cultured in 60-mm dishes; at 30% to 40% confluence, cells were transduced with Ad- EZH2 (adenovirus expressing EZH2 under CMV5 promoter; Vector Biolabs, Malvern, Pennsylvania) and Ad- Green Fluorescent Protein (GFP) at varying multiplicity of infections (0-100 plaque-forming unit/cell), as we have described previously.

Techniques: Over Expression, Plasmid Preparation, Expressing, Infection, RNA Expression, Western Blot

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: BMPC function was directly damaged by 24-h exposure of MGO rather than high glucose (HG) or AGEs. BMPCs from type 2 diabetic db/db mice and the healthy control db/+ mice were cultured in vitro for 7 days. A: Fluorescent staining of Ulex-lectin binding and Dil-ac-LDL uptake and VE-cadherin in 7-day cultured BMPCs (n = 4 per group). Scale bar = 500 μm. B: Flow cytometry analysis of Sca-1, CD34, Flk-1, CD45, and Sca-1/Flk-1 cell surface markers of BMPCs after 7 days of culture (n = 5 per group). BMPCs were then treated with either MGO (10 μmol/L, using H2O as control), AGEs (200 μmol/L, using BSA as control), or HG (25 mmol/L d-glucose, using 5 mmol/L d-glucose plus 20 mmol/L d-mannitol [NG] as control) for 24 or 72 h. C: Schema of MGO and AGE metabolism. MGO as by-product from glucose metabolism can bind to proteins, lipids, and DNA, forming AGEs. GLO1 detoxifies MGO by converting it to d-lactate. D: Dot plot of the percent apoptotic cells in db/+ BMPCs treated with MGO, AGEs, or HG for 24 or 72 h (n = 5 per group). E: Representative scatter plots showing the distribution of Annexin-V+/PI− cells in db/+ BMPCs. F: Network formation quantification in db/+ BMPCs treated with MGO, AGEs, or HG for 24 or 72 h (n = 4 per group). G: Dot plot of migration assay of db/+ BMPCs treated with MGO, AGEs, or HG for 24 or 72 h (n = 5 per group). H: Representative images of tube network formed by db/+ BMPCs treated with MGO, AGEs, or HG for 24 or 72 h. Scale bar = 1,000 μm. I: Representative images of migration assay of db/+ BMPCs treated with MGO, AGEs, or HG for 24 or 72 h. Scale bar = 200 μm. *P < 0.05 vs. controls; **P < 0.01 vs. controls.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Cell Culture, In Vitro, Staining, Binding Assay, Flow Cytometry, Migration

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: Augmentation of GLO1 improved BMPC function, whereas GLO1 deficiency caused BMPC dysfunction. A: GLO1 expression levels in SMCs, BMPCs, and endothelial cells (ECs) (n = 5 per group). **P < 0.01 vs. SMC; #P < 0.05 vs. BMPC. B: Western blot analysis of GLO1 protein in db/+ BMPCs infected with Ad-GLO1, using Ad-GFP as control (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP. C: Dot plot of network formation quantification of db/+ BMPCs infected with Ad-GLO1 or Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP. D: Representative images of network formation in db/+ BMPCs infected with Ad-GLO1 or Ad-GFP (n = 5 per group). Scale bar = 1,000 μm. E: Proliferation of db/+ BMPCs infected with Ad-GLO1 or Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP. F: Western blot analysis of GLO1 expression level in db/+ BMPC transfected with si-GLO1 or scramble oligo (n = 5 per group). *P < 0.05 vs. db/+ plus scramble. G: Network formation in db/+ BMPC transfected with si-GLO1 or scramble oligo (n = 5 per group). *P < 0.05 vs. db/+ plus scramble. H: Representative images of db/+ BMPC transfected with si-GLO1 or scramble oligo. Scale bar = 1,000 μm. M, protein marker.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Expressing, Western Blot, Infection, Transfection, Marker

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: GLO1 overexpression rescued diabetes-induced BMPC dysfunction. A: Western blot analysis showing deficient expression levels of GLO1 in diabetic BMPCs compared with healthy controls (n = 5 per group). *P < 0.01 vs. db/+. B: Western blot analysis of GLO1 protein level in db/db BMPCs infected with Ad-GLO1, using db/db BMPC-infected Ad-GFP as diabetic control (n = 5 per group). *P < 0.05 vs. db/db plus Ad-GFP. C: Dot plot of network formation quantification of db/db BMPCs infected with Ad-GLO1 or Ad-GFP or db/+ BMPCs infected with Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP; #P < 0.05 vs. db/db plus Ad-GFP. D: Representative images of network formation. Scale bar = 1,000 μm. E: Dot plot of proliferation assay of db/db BMPCs infected with Ad-GLO1 or Ad-GFP or db/+ BMPCs infected with Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP. F: Dot plot of migration assay of db/db BMPCs infected with Ad-GLO1 or Ad-GFP or db/+ BMPCs infected with Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP; #P < 0.05 vs. db/db plus Ad-GFP. G: Representative images of migration. Scale bar = 1,000 μm. H: Dot plot of the percent apoptotic cells in db/db BMPCs infected with Ad-GLO1 or Ad-GFP or db/+ BMPCs infected with Ad-GFP (n = 5 per group). *P < 0.05 vs. db/+ plus Ad-GFP; #P < 0.05 vs. db/db plus Ad-GFP. I: Representative scatter plots showing the distribution of Annexin-V+/PI− cells.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Over Expression, Western Blot, Expressing, Infection, Proliferation Assay, Migration

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: Overexpression of GLO1 augments IRE1α expression at the protein level. A: Western blot analysis of IRE1α and the phosphorylated form of IRE1α (p-IRE1α) protein levels after infection with Ad-GLO1 in db/db BMPCs, using db/db BMPCs infected with Ad-GFP as control (n = 5 per group). *P < 0.05 vs. db/db plus Ad-GFP. Representative protein bands are shown. B: Quantitative RT-PCR analysis of Ire1 mRNA level in db/db BMPCs infected with Ad-GLO1 or Ad-GFP using gapdh as internal control (n = 5 per group). C: Luciferase promoter reporter assay of transcriptional activity of IRE1α in db/db BMPCs infected with Ad-GLO1 or Ad-GFP (n = 5 per group). Shown above the dot plot is the structure of LightSwitch Promoter Reporter with precloned oligonucleotides of human IRE1α promoter. D: Western blot analysis of IRE1α expression in GLO1-overexpressed db/db BMPCs transfected with si-IRE1α or scramble oligo, using Ad-GFP–infected and scramble oligo–transfected db/db BMPCs as control (n = 5 per group). *P < 0.05 vs. db/db plus Ad-GFP plus scramble; #P < 0.05 vs. db/db plus Ad-GLO1 plus scramble. E: Western blot analysis of GLO1 expression levels in db/db BMPCs with different gene manipulation (n = 5 per group). *P < 0.05 vs. db/db plus Ad-GFP plus scramble. F: Network formation quantification of GLO1-overexpressed db/db BMPCs transfected with si-IRE1α or scramble oligo, using Ad-GFP–infected and scramble oligo–transfected db/db BMPCs as control (n = 5 per group). *P < 0.05 vs. Ad-GFP plus scramble oligo; #P < 0.05 vs. Ad-GLO1 plus scramble oligo. G: Representative images of network formation. Scale bar = 1,000 μm. H: Western blot analysis of GLO1 and IRE1α protein expression in human PCs from peripheral mononuclear cells in healthy individuals (n = 4) and patients with type 2 diabetes (T2D) (n = 2). Representative bands are shown. M, protein marker.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Over Expression, Expressing, Western Blot, Infection, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Transfection, Marker

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: GLO1 improved diabetic BMPC therapy in wound healing in type 2 diabetic mice. For cell therapies, 1 × 106 db/db BMPCs infected with Ad-GLO1 or Ad-GFP were transplanted onto db/db wounds. A: Wound closure curves in db/db mice that received BMPC therapies (n = 5 per group). B: Representative images of cutaneous wounds taken on days 0, 6, 10, and 16 after wounding. C: Capillary formation as evaluated by CD31 staining (brown) in wounds that received cell therapies. Arrows indicate capillary-like structures at wound edge. Images on right are amplified from the region marked with a square on the left. *P < 0.05 vs. db/db plus Ad-GFP; #P < 0.05 vs. db/+ plus GFP.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Infection, Staining, Amplification

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: Local gene transfer of GLO1 or augmentation of GLO1 by coformulation of tRES- and HESP-improved diabetic wound healing. A: Wound closure curves in db/db mice that received local gene transfer (n = 5 per group). *P < 0.05, **P < 0.01 vs. db/db plus Ad-GFP. B: Capillary formation as evaluated by CD31 staining (brown) in wounds receiving gene transfer. The arrows indicate capillary-like structures at wound edge. Images on the right are amplified from the region marked with a square on the left. C: Representative images of cutaneous wounds. D: GLO1 expression levels in tissue at the edge of wounds in db/db mice that received gene transfer (n = 5 per group). *P < 0.05 vs. db/db plus Ad-GFP. E: Wound closure curves in db/db mice that received tRES-HESP coformulation or vehicle control (0.002% DMSO). *P < 0.05 vs. db/db plus control, **P < 0.01 vs. control. F: Capillary formation as evaluated by CD31 staining (brown) in wounds. The arrows indicate capillary-like structures at wound edge. Images on the right are amplified from the region marked with a box on the left. G: Representative images of cutaneous wounds. H: GLO1 expression level in tissue at the edge of wounds that received tRES-HESP therapy (n = 5 per group). *P < 0.05 vs. control. I: Luciferase promoter reporter assay of transcriptional activity of GLO1 in db/db BMPCs treated with tRES-HESP coformulation showed that tRES-HESP enhanced GLO1 promotor activity (n = 5 per group). *P < 0.05 vs. control. Shown above the dot plot is the structure of LightSwitch Promoter Reporter with precloned oligonucleotides of human GLO1 promoter. M, protein marker.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Staining, Amplification, Expressing, Luciferase, Reporter Assay, Activity Assay, Marker

Journal: Diabetes

Article Title: Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes

doi: 10.2337/db18-0933

Figure Lengend Snippet: GLO1 gene transfer rescued impaired wound healing in IRE1αECKO mice compared with IRE1αflox/flox mice. For local gene transfer, 1 × 108 pfu Ad-GLO1 or Ad-GFP in 40 μL PBS were loaded onto the wounds. A: Wound closure curves in IRE1αECKO mice that received Ad-GLO1 (n = 7) or Ad-GFP (n = 5); IRE1αflox/flox mice that received Ad-GFP (n = 8) were set as controls. *P < 0.05 vs. IRE1αflox/flox plus Ad-GFP. B: Representative images of cutaneous wounds. C: Capillary formation as evaluated by CD31 staining (brown) in wounds that received gene transfer. The arrows indicate capillary-like structures at wound edge. Images on the right are amplified from the region marked with a square on the left. Scale bar = 100 μm. D: Proposed mechanism by which GLO1 improves compromised angiogenesis under diabetic conditions. Elevated blood glucose in patients with diabetes generates excessive MGO. By rapidly reacting with proteins, including IRE1α, MGO causes dysfunction of IRE1α RNase activity, leading to compromised angiogenesis and tissue repair. GLO1, as the main enzyme that metabolizes MGO, can decrease plasma concentrations of MGO, therefore facilitating angiogenesis by partly augmenting functional IRE1α protein levels.

Article Snippet: Transfections of adenovirus carrying human Glo1 gene (Ad-GLO1) (ADV209980; Vector Biolabs), Cre recombinase (Ad-Cre), human IRE1a gene (Ad-IRE1α), or egfp gene (Ad-green fluorescent protein [GFP]) (all three provided by K.Z.) were performed at a multiplicity of infection of 50 for 48 h as described previously ( 27 ).

Techniques: Staining, Amplification, Activity Assay, Functional Assay