Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Prolonged HCMV infection induced endothelial apoptosis. A, HCMV infection resulted in substantial cytopathicity in endothelial cells. HAECs were infected with VHL/E strain of HCMV for 9 days and changes in morphology and viability were observed under light microscope. B, Detection of endothelial apoptosis in HCMV-infected HAECs. VHL/E-infected HAECs (day 9 pi) were stained with anti-IE (HCMV immediate early antigen) antibody to indicate infected cells (red), TUNEL (green) for apoptosis, and DAPI (blue) for nuclei. Merged image shows colocalization. C, Time-dependent apoptosis of HCMV-infected HAECs. HAECs were infected with VHL/E for indicated time periods; cells were stained with anti-IE antibody (red), TUNEL (green), and DAPI (blue). Merged images show colocalization. D, Time-dependent death curves of VHL/E-infected HAECs. TUNEL-positive cells and total cells were counted in 5 fields per coverslip. Results were expressed as the average percentage of TUNEL-positive cells over total cells.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Light Microscopy, Staining, TUNEL Assay

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Activation of the mitochondrial apoptosis pathway in HCMV-infected endothelial cells. A, Increased cleavage of caspases-3 and 9 in HCMV-infected endothelial cells. Total protein from mock, AD169, VHL/E, and Towne strains of infected cells (day 9) was extracted and blotted with anticaspase-9 and anticaspase-3 antibodies. β-Actin was used as an internal sample control. B, Time-dependent increases of expression and cleavage of caspase-3 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibodies against total and cleaved caspase-3. C, Cleavage of caspase-3 in HCMV-infected cells. VHL/ E-Infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-cleaved caspase-3 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, Cleavage of caspase-9 in HCMV-infected cells. VHL/ E-infected HAECs (day 9) were stained anti-IE antibody-FITC (green), anticleaved caspase-9 antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. E, Time-dependent increases in expression and cleavage of caspase-8 in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected cells was extracted and blotted with antibody against caspase-8. F, The involvement of caspases-3, -9, and -8 in HCMV-induced endothelial apoptosis. HAECs were infected with VHL/E for 8 days and treated with 10 μmol/L of caspase inhibitor for 3 days (fresh inhibitors were added daily). TUNEL-positive cells and total cells were counted in 4 fields per coverslip. Results were expressed as the mean±SEM percentage of TUNEL-positive cells over total cells. Using ANOVA, all 3 caspase inhibitors resulted in significantly less apoptosis (P<0.001); the inhibiting effect by caspase-3 or 9 inhibitors was greater than that by caspase-8 (P=0.024).

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Activation Assay, Infection, Expressing, Staining, TUNEL Assay

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Increased Bax and Bak levels in HCMV-infected endothelial cells. A, Increase of Bax and Bak expression in HCMV-infected endothelial cells. Total protein from uninfected (cultured for the same duration) and infected (by AD169, VHL/E, and Towne strains) HAECs (day 9) was extracted and blotted with anti-Bax and anti-Bak antibodies. B, Time-dependent increase of Bax and Bak expression in HCMV-infected cells. After indicated infection time, total protein from mock-infected and VHL/E-infected HAECs was extracted and blotted with anti-Bax and anti-Bak antibodies. C, Increased Bax level in HCMV-infected HAECs. VHL/E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-Bax antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, Increased Bak level in HCMV-infected HAEC. VHL/E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-Bak antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Expressing, Cell Culture, Staining

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: HCMV infection induced p53 phosphorylation in endothelial cells. A, Increase of p53 phosphorylation in HCMV-infected endothelial cells. Total protein from mock-infected and VHL/E-infected HAECs (day 9) was extracted and blotted with anti-phospho p53 (ser15), anti-phospho p53 (ser20), and anti-total p53 antibodies. B, Time-dependent increase in p53 phosphorylation in HCMV-infected cells. HAECs were infected with VHL/E as indicated, and total cell lysates were blotted with antibodies specific for p53 phosphorylated on ser15, ser20, total p53, or β-actin. C, Increased p53 phosphorylation in HCMV-infected HAECs. VHL/ E-infected HAECs (day 9) were stained with anti-IE antibody-FITC (green), anti-phosphor p53 (ser15) antibody-Texas Red (red), and DAPI (blue). Merged image shows colocalization. D, VHL/E-infected HAECs (day 9) were stained with anti-IE (green), anti-phospho p53 (ser 20) (red) antibodies, and DAPI (blue). Merged image shows colocalization.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Staining

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: The involvement of p53 pathway in HCMV-induced endothelial apoptosis. HAECs were infected with VHL/E for 5 days before transfected with different amount of p53 siRNA. Twenty-four hours after transfection, total protein lysates from mock-infected and infected cells were prepared and blotted with antibodies against p53, Bax, Bak, caspase-3, and β-actin.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Infection, Transfection

Journal:

Article Title: Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

doi: 10.1161/01.RES.0000129180.13992.43

Figure Lengend Snippet: Involvement of the ATM/Chk2 pathway in HCMV-induced apoptosis. A, Time-dependent activation of the ATM pathway in HCMV-infected cells. HAECs were infected with VHL/E as indicated, and total cell lysates were blotted with antibodies specific for total ATM, phosphorylated Chk2, and β-actin. B, The involvement of the ATM pathway in HCMV-induced endothelial apoptosis. HAECs were treated with indicated concentrations of caffeine followed by infection with VHL/E. Five days after infection, cell lysates were subjected to immunoblotting with antibodies specific against phosphorylated Chk2, phospho p53 at ser15, and β-actin.

Article Snippet: Infection of HAECs With HCMV Three strains of HCMV (AD169 [ATCC VR-538], Towne [ATCC VR-977], and VHL/E [from W.J.W.]) were used in the study.

Techniques: Activation Assay, Infection, Western Blot

Journal: Cancer Cell International

Article Title: Human cytomegalovirus interleukin-10 enhances matrigel invasion of MDA-MB-231 breast cancer cells

doi: 10.1186/s12935-017-0399-5

Figure Lengend Snippet: MDA-MB-231 cells exhibit increased migration and matrigel invasion in the presence of cmvIL-10. a Transwell migration toward EGF after 5 h in the presence of conditioned medium from mock or HCMV-infected fibroblasts at the indicated MOIs. IL-10R neutralizing antibody (NAb) was included at 30 μg/ml. b Matrigel invasion toward EGF in the presence of 100 ng/ml purified recombinant cmvIL-10 or hIL-10 after 22 h. c Matrigel invasion toward EGF in the presence or absence of 100 ng/ml purified recombinant cmvIL-10 or conditioned medium from mock or infected fibroblasts (MOI = 1). Where indicated, 10 μM Stat3 inhibitor was included. Error bars SEM. *p < 0.05, **p < 0.001. These results are representative of three independent experiments

Article Snippet: Human cytomegalovirus strain AD169 (ATCC) was used to infect confluent monolayers of HFFs at the indicated multiplicities of infection.

Techniques: Migration, Infection, Purification, Recombinant

Journal: Cancer Cell International

Article Title: Human cytomegalovirus interleukin-10 enhances matrigel invasion of MDA-MB-231 breast cancer cells

doi: 10.1186/s12935-017-0399-5

Figure Lengend Snippet: Model depicting possible role of cmvIL-10 in promoting tumor metastasis. A monocyte that is latently infected with HCMV infiltrates a localized tumor, releasing cmvIL-10 that acts on tumor cells expressing the IL-10 receptor. This leads to changes in levels of MTSS1, uPAR and PAI-1, which reduce cell adhesion. Increased levels of uPAR and PAI-1 are strongly associated with increased migration and can also help activate MMP-3. Active MMP-3 degrades proteins in the extracellular matrix, facilitating access for tumor cells to invade surrounding stromal tissue and enter the bloodstream

Article Snippet: Human cytomegalovirus strain AD169 (ATCC) was used to infect confluent monolayers of HFFs at the indicated multiplicities of infection.

Techniques: Infection, Expressing, Migration