Journal: Nature Communications

Article Title: Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

doi: 10.1038/s41467-024-51579-0

Figure Lengend Snippet: a – d Human white preadipocytes were pretreated with vehicle (Veh) or 100 nM EDN3 and with or without inhibitors for 3 days, followed by a 12-day adipogenesis. a Schematic of pretreatment experiments. UCP1 mRNA ( b ) and protein ( c ) levels in mature adipocytes (EV and EV and EDNRB OE). d UCP1 mRNA levels in mature adipocytes (EV and EDNRB KO). e Cytosolic calcium levels in human white preadipocytes after 100 nM EDN3 treatment. f , g Intracellular cAMP levels ( f ) and oxygen consumption rate (OCR; g ) in human white preadipocytes after 6 hours of Veh or EDN3 treatment. Maximal OCR was quantified in the right panel. h – j EV and EDNRB OE preadipocytes were pretreated with Veh or EDN3 combined with or without melittin (ME) or YM254890 (YM) for 3 days, followed by a 12-day adipogenesis. UCP1 mRNA ( h ) and protein ( i ) levels in mature adipocytes pretreated with ME. j UCP1 mRNA levels in mature adipocytes pretreated with YM. k The protein levels in preadipocytes after 6 hr of Veh or EDN3 treatment. The quantification of protein bands was in the right panels. l The EPAC1 protein in EDNRB OE preadipocytes after 48 hr of EPAC1 siRNA treatment. m EDNRB OE preadipocytes with or without EPAC1 knockdown were pretreated with Veh or EDN3 for 3 days, followed by a 12-day adipogenesis. Protein levels in mature adipocytes. n EV or EDNRB OE preadipocytes were pretreated with Veh, 1 μM or 10 μM of PD98059 (PD-1 or PD-10) for 3 days and were differentiated to mature adipocytes. UCP1 and DIO2 mRNA levels in human white adipocytes. n = 3 biological replicates/group in ( b – e ), ( i ), ( k – n ); n = 4 biological replicates/group in ( f – h ), ( j ). Data are presented as mean ± SEM. P values were determined using unpaired two-tailed t tests: ( l ); one-way ANOVA with Tukey’s multiple-comparison test: ( b – d ), ( f – k ), ( m ), ( n ); two-way ANOVA with Bonferroni’s multiple-comparison test: ( e ). Source data are provided as a Source Data file. Panel ( a ) was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Primary antibodies for EDNRB (NBP130599, Novus Biologicals), UCP1 (ab23841, abcam), phospho-ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling), total ERK1/2 (#4695, Cell Signaling), phospho-CREB (Ser133) (#9198, Cell Signaling), total CREB (#9197, Cell Signaling), Phospho-PKA Substrate (#9624, Cell Signaling), PPARγ (GTX32803, GeneTex), FABP4/AP2 (GTX636995, GeneTex), EPAC1 (#4155, Cell Signaling), TUBULIN (#2146, Cell Signaling), VINCULIN (sc-25336, Santa Cruz) or ACTIN (MAB1501, Millipore) were applied in blocking buffer over night at 4°C.

Techniques: Knockdown, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

doi: 10.1038/s41467-024-51579-0

Figure Lengend Snippet: a , b Male mice were intraperitoneally injected by tamoxifen 5 times within 7 days to induce EDNRB KO in PDGFRα + preadipocytes (iKO) and were then housed at 5 °C for 7 days. a The mRNA levels of thermogenic genes in scWAT. CTL: n = 5, iKO: n = 6. b The protein levels of UCP1 in scWAT. The quantification of protein bands in right panel. n = 4 mice/group. c , d Control and iKO mice were housed at thermoneutral temperature (30 °C, TN) or cold (5 °C) for 7 days. Images of immunohistochemical stain for UCP1 proteins (UCP1 IHC; c ) and HE stain ( d ) in scWAT. High magnification images were in the right panels. Scale bar=100 μm. Two independent experiments were repeated with similar results, as shown in Supplementary Fig. a and . e The phosphorylated ERK and total ERK protein in scWAT of mice after 7 days of cold exposure. The quantification of protein bands in right panel. n = 3 mice/group. f , g PDGFRα + cell lineage-tracing control and EDNRB iKO mice were housed at thermoneutral temperature (30 °C, TN) or cold (5°C) for 7 days. f The immunofluorescent images from the section of scWAT. Scale bar=100 μm. g Quantification of GFP signal in each section of scWAT. CTL at TN: n = 6, iKO at TN: n = 6, CTL at Cold: n = 6, iKO at Cold: n = 7, 4 slides/mouse. h The mRNA levels of Ednrb, Pdgfrα and Ebf2 in SVF of scWAT from mice housed at TN or cold for 7 days. CTL at TN: n = 3, iKO at TN: n = 3, CTL at Cold: n = 3, iKO at Cold: n = 4. Data are presented as mean ± SEM. P values were determined using unpaired two-tailed t tests: ( a) , ( b ), ( e ); one-way ANOVA with Tukey’s multiple-comparison test: ( g ), ( h ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for EDNRB (NBP130599, Novus Biologicals), UCP1 (ab23841, abcam), phospho-ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling), total ERK1/2 (#4695, Cell Signaling), phospho-CREB (Ser133) (#9198, Cell Signaling), total CREB (#9197, Cell Signaling), Phospho-PKA Substrate (#9624, Cell Signaling), PPARγ (GTX32803, GeneTex), FABP4/AP2 (GTX636995, GeneTex), EPAC1 (#4155, Cell Signaling), TUBULIN (#2146, Cell Signaling), VINCULIN (sc-25336, Santa Cruz) or ACTIN (MAB1501, Millipore) were applied in blocking buffer over night at 4°C.

Techniques: Injection, Control, Immunohistochemical staining, Staining, H&E Stain, Two Tailed Test, Comparison

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: EDNRB is identified as a key downstream target of REG Iα. Principal Component Analysis (PCA) showed sample distribution. (B) Violin plots showed the overall FPKM distribution. (C) A volcano plot showed differentially expressed genes. (D) KEGG pathway analysis revealed enrichment in the calcium signaling pathway. Pathway annotations were performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( www.kegg.jp ). (E) Expression of selected genes, including EDNRB, in LoVo cells was validated by RT‐qPCR. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: REG Iα regulates cell proliferation, apoptosis, and Ca 2+ signaling via EDNRB. (A) qPCR showed REG Iα and EDNRB expression. (B) REG Iα, EDNRB, and p‐CaMKII protein levels were detected by Western blot. β‐actin served as the loading control. (C) Intracellular Ca 2+ concentration was measured using Fluo‐4 AM probe. (D) EdU assay assessed cell proliferation. (E) Cell viability was measured by CCK‐8 assay. (F) Cell apoptosis was detected by flow cytometry. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Expressing, Western Blot, Control, Concentration Assay, EdU Assay, CCK-8 Assay, Flow Cytometry

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: REG Iα promotes cell migration, invasion, and EMT via EDNRB. (A) Cell migration was assessed by Transwell assay. (B) Matrigel‐coated Transwell assay assessed cell invasion. (C) Western blot analysis of EMT‐related markers, including the epithelial marker E‐Cadherin and mesenchymal markers N‐Cadherin and Vimentin. β‐actin was used as the loading control. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Migration, Transwell Assay, Western Blot, Marker, Control

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: The REG Iα‐EDNRB axis promotes cell proliferation and survival via the Ca 2+ signaling pathway. REG Iα, EDNRB, and p‐CaMKII protein levels were detected by Western blot. β‐actin was used as the loading control. (B) Intracellular Ca 2+ concentration was measured using Fluo‐4 AM probe. (C) EdU assay assessed cell proliferation. (D) Cell viability was measured by CCK‐8 assay. (E) Cell apoptosis was detected by flow cytometry. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Western Blot, Control, Concentration Assay, EdU Assay, CCK-8 Assay, Flow Cytometry

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: The REG Iα‐EDNRB axis suppresses ER stress‐induced apoptosis and ROS accumulation. (A) Western blot analysis of ER stress markers (GRP78, CHOP) and apoptotic executioners (Cleaved Caspase‐3, Cleaved PARP) in CRC cells under the indicated treatment conditions. β‐Actin was used as the loading control. (B) Intracellular ROS levels detected by DCFH‐DA staining. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Western Blot, Control, Staining

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: The REG Iα‐EDNRB axis promotes cell migration, invasion, and EMT via the Ca 2+ signaling pathway. (A) Transwell assay assessed cell migration. (B) Cell invasion was assessed by Matrigel‐coated Transwell assay. (C) EMT‐related protein levels (E‐Cadherin, N‐Cadherin, Vimentin) were detected by Western blot. β‐actin served as the loading control. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: Migration, Transwell Assay, Western Blot, Control

Journal: Cancer Medicine

Article Title: Role of Ca 2+ ‐Dependent Epithelial‐Mesenchymal Transition in Malignant Progression of Colorectal Cancer: Special Focus on REG Iα/ EDNRB

doi: 10.1002/cam4.71754

Figure Lengend Snippet: The REG Iα‐EDNRB‐Ca 2+ axis promotes tumor growth and EMT in vivo. (A) Representative images of the xenograft tumors and excised tumor tissues from the indicated groups. (B) Statistical analysis of the final tumor weights. (C) Tumor growth curves measuring tumor volume over time. (D) H&E staining showed pathological changes in tumor tissues. (E) TUNEL assay detected cell apoptosis in tumor tissues. (F) Cell proliferation in tumor tissues was shown by Ki67 IHC staining. (G) Expression of REG Iα (Immunofluorescence, upper row) and EDNRB (IHC, lower row) in tumor tissues. (H) Western blot analysis of REG Iα, EDNRB, and p‐CaMKII protein levels in tumor tissues. β‐actin was used as the loading control. (I) Western blot analysis of EMT‐related proteins (E‐Cadherin, N‐Cadherin, Vimentin) in tumor tissues. β‐actin was used as the loading control. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking with 5% skimmed milk for 1.5 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against the following targets: REG Iα (Abcam, ab231092), EDNRB (BOSTER, PB9554), p‐CaMKII (Abcam, ab124880), GRP78(BOSTER, BA4293).

Techniques: In Vivo, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Immunofluorescence, Western Blot, Control

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