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. 2015 Dec 24;2(4):ofv155. doi: 10.1093/ofid/ofv155

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© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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Figure 3. — Human cytomegalovirus (HCMV) infection alters endothelin receptor type B (ET_BR) cellular distribution, induces surface expression of ET_BR and its effect on endothelin receptor type A (ET_AR). (A) Representative immunofluorescence staining on HCMV-infected endothelial cells at 3 days postinfection to illustrate the subcellular distribution of ET_BR in uninfected and infected cells. Arrow shows increased ET_BR protein expression in the perinuclear region. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of ET_BR 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_BR 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_BR at indicated days postinfection (dpi). (C) The effects of different multiplicity of infection (moi) of HCMV on ET_BR 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_BR (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_BR 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_BR surface expression in SMCs, hPASMCs (left column) and hSMCs (right column) at 5 dpi. Y-axis denotes log intensity of ET_BR (left panel), whereas x-axis is SS Lin. The effects of HCMV on ET_AR 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).

Figure 3. — Human cytomegalovirus (HCMV) infection alters endothelin receptor type B (ET_BR) cellular distribution, induces surface expression of ET_BR and its effect on endothelin receptor type A (ET_AR). (A) Representative immunofluorescence staining on HCMV-infected endothelial cells at 3 days postinfection to illustrate the subcellular distribution of ET_BR in uninfected and infected cells. Arrow shows increased ET_BR protein expression in the perinuclear region. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of ET_BR 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_BR 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_BR at indicated days postinfection (dpi). (C) The effects of different multiplicity of infection (moi) of HCMV on ET_BR 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_BR (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_BR 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_BR surface expression in SMCs, hPASMCs (left column) and hSMCs (right column) at 5 dpi. Y-axis denotes log intensity of ET_BR (left panel), whereas x-axis is SS Lin. The effects of HCMV on ET_AR 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).