atcc vr 977d  (ATCC)

Journal: PLOS Pathogens

Article Title: EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells

doi: 10.1371/journal.ppat.1011304

Figure Lengend Snippet: A , U138 cells were transfected with siRNA pools targeting the Eph receptors family for 36 h. Then cells were infected with HCMV for 3 days, and HCMV-positive cells were quantified by flow cytometry. Bars represent the percentage of infection determined by flow cytometry, with infection of control siRNA duplex (siCtrl) transfected cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent three independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. NS, not significant; ** P <0.01; *** P < 0.001.

Article Snippet: Each siRNA pool was transfected into U138 individually for 48 h, then infected with a reconstructed HCMV Towne strain (ATCC-VR977) expressing GFP [ , ].

Techniques: Transfection, Infection, Flow Cytometry, Control

Journal: PLOS Pathogens

Article Title: EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells

doi: 10.1371/journal.ppat.1011304

Figure Lengend Snippet: A to D , The U138 cells (A) and U251 cells (C) were transfected with EphA2 siRNAs (siA2-1#, siA2-2#, siA2-3#) or siCtrl for 36 h. Part of the cells was harvested, and their EphA2 protein level was analyzed by WB, using α-tubulin as a loading control (representative of 3 independent experiments). The remaining cells were infected with HCMV and HCMV-positive cells and then were analyzed by flow cytometry (B and D). Bars represent the percentage of infection determined by flow cytometry, with infection of siCtrl transfected cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001. E, F , sgRNAs targeting EphA2 (sgA2-1#, sgA2-2#) were delivered into U138 cells by the lentivirus package system. Cells were selected by puromycin for 3 days. The empty vector was used as control (sgVector). Part of the cells was harvested, and their EphA2 protein level was analyzed by WB (E), using α-tubulin as a loading control (representative of three independent experiments). The remaining cells were infected with HCMV and HCMV-positive cells and then were analyzed by flow cytometry (F). Bars represent the percentage of infection determined by flow cytometry, with infection of sgVector transduced cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001. G, H , The plasmid expressing Flag-tagged EphA2, EphA2 Δcyto or empty vector was stably transduced into T98G cells. Cells were harvested, and the EphA2 expression was analyzed by WB (G). α-tubulin was used as a loading control. Data are representative of 3 independent experiments. HCMV was used to infect these cells, and the HCMV infection efficiency was analyzed by flow cytometry (H). Bars represent the percentage of infection, with infection of empty vector-transfected cells normalized to 1. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001.

Article Snippet: Each siRNA pool was transfected into U138 individually for 48 h, then infected with a reconstructed HCMV Towne strain (ATCC-VR977) expressing GFP [ , ].

Techniques: Transfection, Control, Infection, Flow Cytometry, Plasmid Preparation, Expressing, Stable Transfection

Journal: PLOS Pathogens

Article Title: EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells

doi: 10.1371/journal.ppat.1011304

Figure Lengend Snippet: A , U138 cells were infected with HCMV in the presence of an indicated concentration of 2,5-dimethylpyrrolyl benzoic acid derivative. HCMV infection efficiency was quantified by flow cytometry after 3 days. Bars represent the percentage of infection determined by flow cytometry, with infection of no 2,5-dimethylpyrrolyl benzoic acid derivative treated cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001. B , U138 cells were infected with HCMV in the presence of an indicated concentration of EphA2 antibody or rabbit IgG control. HCMV infection efficiency was quantified by flow cytometry after 3 days. Bars represent the percentage of infection determined by flow cytometry, with infection of rabbit IgG control-treated cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001. C, Glioblastoma organoids were stained with hematoxylin-eosin staining (HE) and EphA2 antibody. Images of insets were magnified 3 times. Scale bars: 100 μm. Representative images from the samples were detected. D, E , Glioblastoma organoids were infected with HCMV in the presence of an indicated concentration of 2,5-dimethylpyrrolyl benzoic acid derivative. HCMV genome DNA was extracted from HCMV-infected organoids, and the copy number of HCMV was measured using qPCR. The GAPDH DNA was used for cell counting estimation (D). Bars represent relative HCMV DNA copy number determined by qPCR, with the copy number of no 2,5-dimethylpyrrolyl benzoic acid derivative treated cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 2 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001. HCMV-positive cells were shown green in representative fluorescence images after 3 days (E).

Article Snippet: Each siRNA pool was transfected into U138 individually for 48 h, then infected with a reconstructed HCMV Towne strain (ATCC-VR977) expressing GFP [ , ].

Techniques: Infection, Concentration Assay, Flow Cytometry, Control, Staining, Cell Counting, Fluorescence

Journal: PLOS Pathogens

Article Title: EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells

doi: 10.1371/journal.ppat.1011304

Figure Lengend Snippet: A , The T98G cells stably expressing EphA2 were incubated with HCMV on ice for 2 h and then 30 minutes at 37°C. Cells were washed with Hanks solution three times. The surface-bound virus was removed by proteinase K digestion. Then HCMV DNA copy number per cell was measured by qPCR. Bars represent the percentage of HCMV entry, with the entry of empty vector-transfected cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 2 independent experiments, two-tailed unpaired Student’s t -test. *** P < 0.001. B , EphA2 knockout cells were incubated with HCMV on ice for 2 h and then 30 minutes at 37°C. Cells were washed with Hanks solution three times. The surface-bound virus was removed by proteinase K digestion. Then HCMV DNA copy number per cell was measured by qPCR. Bars represent the percentage of HCMV entry, with the entry of sgVector transfected cells normalized to 100%. Data are mean ± s.e.m. (n = 3 biological replicates) and represent 2 independent experiments. One-way ANOVA was carried out with Dunnett’scorrection for multiple comparisons. *** P < 0.001. C , Effector HEK-293T cells were transfected with pT7-EMCLuc, pRL-SV40 and gB or gH/gL or gO or gH/gL/gB or gH/gL/gO/gB. Cells then were co-cultured with HEK-293T cells transfected with a plasmid for T7 polymerase. The relative fusion activity was calculated as the ratio of firefly to Renilla luciferase activity after 24 h. Data are mean ± s.e.m. (n = 4 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. NS P >0.05, *** P < 0.001. D , Cell-based HCMV fusion assay by co-culturing HEK-293T cells transfected with plasmids expressing EphA2 or vector and HEK-293T transfected with gB or gH/gL or gO or gH/gL/gB or gH/gL/gO/gB. Bars represent the percentage of fusion, with the fusion of vector-transfected cells normalized to 100%. Data are mean ± s.e.m. (n = 4 biological replicates) and represent 2 independent experiments, One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. NS P >0.05, ** P < 0.01, *** P < 0.001. E , Effector HEK-293T cells were transfected with pT7-EMCLuc, pRL-SV40 and gH/gL/gO/gB. Cells then were co-cultured with HEK-293T cells transfected with a plasmid for T7 polymerase in the presence of an indicated concentration of EphA2 antibody or rabbit IgG control. The relative fusion activity was calculated as the ratio of firefly to Renilla luciferase activity after 24 h. Data are mean ± s.e.m. (n = 4 biological replicates) and represent 3 independent experiments. One-way ANOVA was carried out with Dunnett’s correction for multiple comparisons. *** P < 0.001.

Article Snippet: Each siRNA pool was transfected into U138 individually for 48 h, then infected with a reconstructed HCMV Towne strain (ATCC-VR977) expressing GFP [ , ].

Techniques: Stable Transfection, Expressing, Incubation, Virus, Plasmid Preparation, Transfection, Two Tailed Test, Knock-Out, Cell Culture, Activity Assay, Luciferase, Single Vesicle Fusion Assay, Concentration Assay, Control