Journal: BMC Neuroscience

Article Title: Upregulation of the endothelin A (ET A ) receptor and its association with neurodegeneration in a rodent model of glaucoma

doi: 10.1186/s12868-017-0346-3

Figure Lengend Snippet: Overexpression of the ET A receptor can increase in ET B receptor expression. a Representative images. Immunoblot showing the expression of ET A and ET B receptors in the plasma membrane fraction of 661W stable clones overexpressing the ET A receptor. b Immunoblot densitometry showing fold change in protein expression of both ET A and ET B receptors in 661W cells overexpressing the ET A receptor compared to empty vector cells. Protein normalized to calnexin expression. Bars represent mean ± SEM (n = 5). Asterisks indicated statistical significance *p < 0.05 by student’s t-test

Article Snippet: Primary antibodies used to probe blots were rabbit anti-ET A (1:1000; Sigma), rabbit anti-ET B (1:10,000, Antibody Research Corporation), rabbit anti-Calnexin (1:1000, Cell Signaling) and mouse calnexin (1:1000, Cell Signaling).

Techniques: Over Expression, Expressing, Western Blot, Clone Assay, Plasmid Preparation

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Effects of human cytomegalovirus (HCMV) on vascular endothelial and smooth muscle cells. The graphic depicts relative expression of endothelin receptor type B (ET B R) with indicated multiplicity of infection (moi) of HCMV as assayed by quantitative TaqMan polymerase chain reaction (PCR) at indicated days postinfection (dpi) for endothelial cells (human umbilical vein cells [HUVECs]; A.i) or smooth muscle cells (SMCs) (human pulmonary arterial SMCs [hPASMCs], A.ii) and human aortic SMCs [hSMCs], A.iii). The corresponding infection levels in HCMV-infected HUVECs (A.iv), hPASMCs (A.v) and hSMCs (A.vi), as assessed by quantitative TaqMan PCR are shown. The effects of UV-irradiated HCMV on the ET B R mRNA level in hPASMC (A.vii) and hSMC (A.viii) are illustrated. The uninfected cells at the indicated time course were used as a calibrator for relative quantification using the comparative 2 −ΔΔCt method with β 2 -microglobulin as an endogenous control (biological replicates for A.i-A.vi [n = 4] and A.vii and A.viii [n = 3] with technical duplicates for each PCR; values are mean ± standard deviation [SD]). Representative immunofluorescence staining on HCMV-infected endothelial cells (HUVECs; B.i) and SMCs (hPASMC, B.ii and hSMC, B.iii) with indicated MOI at 3 dpi. Arrows in B.i-B.iii depict HCMV-infected cells without up-regulation of ET B R. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI], blue), and the immunoreactivity of ET B R or HCMV immediate-early (IE) was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively (biological replicates, n = 3). The corresponding quantitative results are shown for HCMV-infected HUVECs (B.iv), hPASMC (B.v) and hSMC (B.vi), by mean florescent intensity expressed as pixel sum as analyzed with Leica Application Suite Advanced Fluorescence software. At least 5 different areas of interest were quantified (values are mean ± SD; P < .05 was considered to be statistically significant; * P = .01–.05; ** P = .001–.01; *** P < .001; n.s = not significant).

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-HCMV-IE (1:500), rabbit anti-ET B R (1:500), mouse anti-giantin (1:300) (clone G1/133, marker for Golgi; Enzo Life Sciences), and endoplasmic reticulum marker PDI (1:300) (clone 1D3; Enzo Life Sciences), with secondary antibody goat anti-mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 594 (both from the Molecular Probes and used at 1:500 dilution).

Techniques: Expressing, Infection, Polymerase Chain Reaction, Irradiation, Standard Deviation, Immunofluorescence, Staining, Fluorescence, Software

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Human cytomegalovirus (HCMV) immediate-early or early gene is responsible for endothelin receptor type B (ET B R) up-regulation. Relative expression of ET B R in uninfected or HCMV-infected cells (human umbilical vein cells) treated with different concentrations of ganciclovir and analyzed at 5 days postinfection (values are mean ± standard deviation, biological replicates, n = 2 with technical duplicates for each polymerase chain reaction; ** P = .001–.01; *** P < .001; Abbreviation: n.s = not significant).

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-HCMV-IE (1:500), rabbit anti-ET B R (1:500), mouse anti-giantin (1:300) (clone G1/133, marker for Golgi; Enzo Life Sciences), and endoplasmic reticulum marker PDI (1:300) (clone 1D3; Enzo Life Sciences), with secondary antibody goat anti-mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 594 (both from the Molecular Probes and used at 1:500 dilution).

Techniques: Expressing, Infection, Standard Deviation, Polymerase Chain Reaction

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Human cytomegalovirus (HCMV) infection alters endothelin receptor type B (ET B R) cellular distribution, induces surface expression of ET B R and its effect on endothelin receptor type A (ET A R). (A) Representative immunofluorescence staining on HCMV-infected endothelial cells at 3 days postinfection to illustrate the subcellular distribution of ET B R in uninfected and infected cells. Arrow shows increased ET B R protein expression in the perinuclear region. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of ET B R or HCMV immediate-early (IE) was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively (biological replicates, n = 3). (B) Representative immunofluorescence staining of ET B R and giantin, a Golgi marker (top panel), or PDI, an endoplasmic reticulum marker (bottom panel). Nucleus was stained with DAPI (blue). Flow cytometry analysis on the effects of HCMV-infected endothelial cells (human umbilical vein cells [HUVECs]) or smooth muscle cells (SMCs) (human pulmonary arterial SMCs [hPASMCs] and human aortic SMCs [hSMCs]) on ET B R at indicated days postinfection (dpi). (C) The effects of different multiplicity of infection (moi) of HCMV on ET B R surface expression at 1, 3, and 5 dpi (left panel) in HUVECs. The HCMV IE is shown on the right panel. Y-axis denotes log intensity of ET B R (left panel) or IE (right panel), whereas x-axis is side scatter in linear scale (SS Lin). (D) The effects of ultraviolet (UV)-irradiated HCMV on ET B R surface expression in HUVECs. Little or no IE was detected with the UV treatment (bottom row). (E) The effects of UV-irradiated HCMV (middle row) or HCMV (bottom row) on ET B R surface expression in SMCs, hPASMCs (left column) and hSMCs (right column) at 5 dpi. Y-axis denotes log intensity of ET B R (left panel), whereas x-axis is SS Lin. The effects of HCMV on ET A R mRNA level in smooth muscle cells, hPASMC (F) and hSMC (G) (MOI; biological replicates, n = 3 with technical duplicates for each polymerase chain reaction; values are mean ± standard deviation; P < .05 was considered to be statistically significant; * P = .01–.05; ** P = .001–.01; *** P < .001).

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-HCMV-IE (1:500), rabbit anti-ET B R (1:500), mouse anti-giantin (1:300) (clone G1/133, marker for Golgi; Enzo Life Sciences), and endoplasmic reticulum marker PDI (1:300) (clone 1D3; Enzo Life Sciences), with secondary antibody goat anti-mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 594 (both from the Molecular Probes and used at 1:500 dilution).

Techniques: Infection, Expressing, Immunofluorescence, Staining, Marker, Flow Cytometry, Irradiation, Polymerase Chain Reaction, Standard Deviation

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Human cytomegalovirus (HCMV) antigens are closely associated with endothelin receptor type B (ET B R) in human carotid atherosclerotic plaques. Representative photomicrographs of immunohistochemistry and immunofluorescence (IF) staining on frozen human carotid atherosclerotic plaques. (A) Immunohistochemistry staining to show colocalization of HCMV immediate-early (IE) and ET B R in Von Willebrand factor (vWF)-positive cells as revealed by chromogen diaminobenzidine, brown products in a boxed area with consecutive sections (left column; right column is a higher magnification of the boxed areas). (B) The IF staining on a human carotid atherosclerotic plaque to illustrate the colocalization of HCMV-IE with ET B R in fibroblast-like cells. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of HCMV-IE or ET B R was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively. Twenty human carotid atherosclerotic plaques were examined.

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-HCMV-IE (1:500), rabbit anti-ET B R (1:500), mouse anti-giantin (1:300) (clone G1/133, marker for Golgi; Enzo Life Sciences), and endoplasmic reticulum marker PDI (1:300) (clone 1D3; Enzo Life Sciences), with secondary antibody goat anti-mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 594 (both from the Molecular Probes and used at 1:500 dilution).

Techniques: Immunohistochemistry, Immunofluorescence, Staining

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: The effects of human cytomegalovirus (HCMV) and endothelin receptor type B (ET B R) blocker, BQ788, on endothelial cells proliferation, migration, vasculogenecity, and leukocyte recruitment. (A) Cell proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was performed in human umbilical vein cells (HUVECs) uninfected or HCMV-infected, untreated or treated with BQ788 at the indicated time points after infection. Optical density was read at 590 nm with reference wavelength at 670 nm. (B) Light microscopic images (×4) of the wound healing assay were taken at different times after the “scratch” of HUVEC monolayers uninfected or HCMV-infected treated with BQ788. (C) Light microscopic images (×4) of tube formation by HUVEC uninfected or HCMV-infected and treated with BQ788. (D) Light microscopic images of flow adhesion assay in cells stimulated with tumor necrosis factor-α (TNFα) and uninfected or HCMV-infected (left panel). The number of adherent cells was quantified for all the conditions (right panel). Values are mean ± standard deviation; P < .05 was considered to be statistically significant; * P = .01–.05; ** P = .001–.01; *** P < .001).

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-HCMV-IE (1:500), rabbit anti-ET B R (1:500), mouse anti-giantin (1:300) (clone G1/133, marker for Golgi; Enzo Life Sciences), and endoplasmic reticulum marker PDI (1:300) (clone 1D3; Enzo Life Sciences), with secondary antibody goat anti-mouse or goat anti-rabbit conjugated to Alexa Fluor 488 or 594 (both from the Molecular Probes and used at 1:500 dilution).

Techniques: Migration, Proliferation Assay, Infection, Wound Healing Assay, Cell Adhesion Assay, Standard Deviation