Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effects of α 1 -AR stimulation on the expression of mitochondrial energetic molecules, oxidative phosphorylation, and biological functions in skeletal and cardiac muscle cells and liver cells. (a, b) The expression of p-AMPK and PPAR δ in C2C12, HL1, and HepG2 cells was stimulated with 1–30 μ M midodrine for the indicated times. (c) Cytosolic calcium mobilization after midodrine treatment in C2C12 and HL1 cells. Each cell type was pretreated with the calcium reactive dye Fluo-3 AM for 45 min and then stimulated with 30 μ M midodrine for the indicated times. Green fluorescence emitted by Fluo-3 AM was detected using confocal microscopy. (d) The phosphorylation of AMPK α at Thr172 and expression of PPAR δ in C2C12 and HL1 cells after pretreatment with the calcium/calmodulin-dependent protein kinase kinase antagonist STO-609 for 25 min and treatment with midodrine. (e) Fluorescence after using the CytoPainter mitochondrial staining kit in midodrine-treated and control C2C12 cells. Original magnification was 200x. (f) The measured activity of succinate dehydrogenase (SDH) in C2C12 cells. (G) Oxygen consumption rate (OCR) in C2C12 cells treated with midodrine (30 μ M), as measured by a Seahorse XFp analyzer. (h) ATP content in C2C12 cells treated with midodrine (30 μ M) cultured with low-glucose (5.56 mM) medium. (i) Glucose transporter (GLUT) 4 protein expression in C2C12 cells treated with high glucose (HG) and midodrine (HG+Mido), HG and insulin (HG+Insulin), and the control treatment (Ctrl). (j) The uptake of 2-deoxyglucose in C2C12 skeletal muscle cells treated with midodrine. (k) OCR (measured by the Seahorse XFp analyzer) in H9C2 cells treated with midodrine (30 μ M) and cultured with low-glucose (5.56 mM) medium. (l) ATP content in H9C2 cells treated with midodrine (30 μ M). Data are expressed as the mean ± standard deviation of triplicate experiments. AMPK: adenosine monophosphate-activated protein kinase; p-AMPK: phosphorylated AMPK; PPAR δ : peroxisome proliferator-activated receptor delta; PGC-1 α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mGLUT4: GLUT4 expression of the cell membrane; tGLUT4: total cellular expression of GLUT4; Ctrl: an untreated control group; Mido: midodrine-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Phospho-proteomics, Fluorescence, Confocal Microscopy, Staining, Control, Activity Assay, Cell Culture, Standard Deviation, Membrane

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effect of midodrine on the endothelial expression of p-AMPK and p-eNOS in HUVECs; OCR analyses in H9C2 cells; intracellular fat and the expression of PPAR δ , p-AMPK, and PGC-1 α in differentiated 3T3-L1 cells; and the effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. (a) The expression of phosphorylated AMPK (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS) proteins in human umbilical vein endothelial cells (HUVECs) treated with cholesterol and palmitate, and the effects from the addition of GSK0660, a PPAR δ antagonist. Ctrl: the control group; CP: the cholesterol- and palmitate-treated group; CPM: the cholesterol-, palmitate-, and midodrine-treated group. (b) The maximal oxygen consumption rate (OCR) analysis as estimated using a Seahorse XFp analyzer and ATP content measured by ELISA in H9C2 cells. (c) The effect of compound C (1 μ M) on p-AMPK expression and PPAR δ expression. (d) The effect of midodrine on intracellular lipid deposits (Oil Red O staining result) and the protein levels of PPAR δ , AMPK, and PGC-1 α in differentiated 3T3-L1 cells treated with midodrine and GSK0660. (e) The effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. Ctrl: untreated control group; Mido: midodrine-treated group; Mido+GSK0660: midodrine- and GSK0660-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HUVEC (Normal Primary Human Umbilical Vein Endothelial Cells) , Lifeline Cell Technology , Cat. #FC-0003 , https://www.lifelinecelltech.com/shop/cells/human-endothelial-cells/umbilical-vein-endothelial-cells/huvec-fc-0003/.

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay

Journal: Redox Biology

Article Title: Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells

doi: 10.1016/j.redox.2020.101816

Figure Lengend Snippet: Induction of Nrf2 signaling by USS depends on SIA integrity. ( A ) Representative immunoblot of HO-1 expression in HUVEC subjected to USS (15 dyn cm -2 ), OSS (±5 dyn cm -2 , 1 Hz) or maintained in static culture for 48 h. Densitometric analysis of HO-1 levels is shown relative to β-actin. Data are mean ± S.E.M. (n = 3 different donors). *P < 0.05; **P < 0.01; *** P < 0.001 (1-way ANOVA). ( B ) EA. hy926 cells stably transduced with lentiviral particles containing Nrf2 silencing shRNA (LvNrf2), scrambled sequences (Scr) or left untransfected (Control) were exposed to USS (15 dyn cm -2 ) for 24 h. HO-1 expression was assessed by immunoblotting relative to β-actin. Data denote mean ± S.E.M. (n = 3 independent experiments). *P < 0.05 (2-way ANOVA). ( C–D ) Following SIA removal with neuraminidase (Neur., 2 U ml -1 , 30 min), both control and Neur.-treated HUVEC cultures were exposed to USS (15 dyn cm -2 ) for 6, 8 and 24 h or immediately terminated (0 h), as indicated. ( C ) Representative confocal images of SIA stained with WGA-CF TM 448A (red) in fixed HUVEC. WGA mean cell intensity (MCI) was quantified in the x-y optical slices and normalized to the number of cell nuclei stained with DAPI (blue). Data denote mean ± S.E.M. (n = 3–6 different donors). *P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA). Scale bar= 20 μm. ( D ). Protein expression and densitometric analyses of the Nrf2 targets HO-1, NQO1 and GCLM relative to β-actin. Data denote mean ± S.E.M. (n = 4–5 different donors). *P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The HUVEC-derived endothelial cell line EA. hy926 (gifted by Unilever UK) [ ] was used for infection with lentiviral vectors and maintained under the same conditions as HUVEC.

Techniques: Western Blot, Expressing, Stable Transfection, Transduction, shRNA, Control, Staining

Journal: Redox Biology

Article Title: Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells

doi: 10.1016/j.redox.2020.101816

Figure Lengend Snippet: Endogenous NEU1 knockdown enhances Nrf2-mediated antioxidant signaling. EA. hy926 cells transduced with lentiviral particles either containing sialidase 1 (NEU1) silencing shRNA (LvNEU1) or scrambled sequences (Scr) were exposed to USS (15 dyn cm -2 ), OSS (±5 dyn cm -2 , 1 Hz) or maintained in static culture for 48 h. ( A ) Representative immunoblot and densitometric analysis of NEU1 expression in whole cell lysates presented relative to β-actin. Data denote mean ± S.E.M. (n = 5 independent cultures). *P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA). ( B ) Representative fluorescence images of the SIA component of the GCX stained with WGA-CF TM 448A (red) in fixed cells using an inverted epi-fluorescence microscope. WGA mean cell intensity (MCI) was normalized to the respective number of cell nuclei stained with DAPI (blue). Data are expressed as mean ± S.E.M. (n = 3 independent cultures). Scale bar= 20 μm . ( C ) Representative immunoblots and densitometric analyses of HO-1, NQO1 and GCLM expression in whole cell lysates presented relative to β-actin. Data denote mean ± S.E.M. (n = 5 independent cultures). *P < 0.05; **P < 0.01; ***P < 0.001 (2-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The HUVEC-derived endothelial cell line EA. hy926 (gifted by Unilever UK) [ ] was used for infection with lentiviral vectors and maintained under the same conditions as HUVEC.

Techniques: Knockdown, Transduction, shRNA, Western Blot, Expressing, Fluorescence, Staining, Microscopy