Figure 8 E, F

A. THP-1 cells were infected by spinoculation with an MOI of 20 with either nonfluorescent TB40/E, UL88-STOP, or UL88-Rev, the latter two of which express GFP driven from the IE2 promoter or were left uninfected. GFP fluorescence was measured by flow cytometry at d5 post infection. B. Schematic representation of the experiment methodology for C and D. At day 3, 2.5 x 105 THP-1, of which ~ 2.5 x 104 were HCMV-infected, were transferred onto a monolayer of ~ 2.5 x 105 HDFs and virus spread was monitored every three days by confocal imaging up to day 15. C. Representative images of TB40/E-GFP and UL88-STOP from day 3 and day 7 are shown. D. The spread of infection was quantified as the area of fluorescence using the NIS element software. Graph shows fold change in area of fluorescence for each virus. E. MRC-5 cells were infected with TB40/E-mCh or UL88-STOP-mCh virus at MOI of 0.05. Cells were harvested for RNA isolation at 7 dpi. Antiviral gene expression was profiled using a set of 84 ISGs by qPCR. Results are plotted as volcano plot depicting the modulated genes as compared between TB40/E WT HCMV and UL88-STOP HCMV infection. F. ISGs known to be modulated in HCMV infection are shown with their relative fold change in mRNA levels (normalized to GAPDH) between TB40/E WT and UL88-STOP virus infection. The graph shows the genes that remained unchanged (green box), downregulated (red box), or upregulated (blue box), in TB40/E WT vs UL88-STOP HCMV-infected cells. Results are from biological triplicates.

A. 以感染复数（Multiplicity of Infection, MOI）为20的剂量，通过离心介导感染法（spinoculation），分别用非荧光型TB40/E、UL88-STOP或UL88-Rev感染THP-1细胞；其中后两种病毒携带由IE2启动子（IE2 promoter）驱动表达的绿色荧光蛋白（GFP），同时设置未感染对照组。于感染后第5天（d5）采用流式细胞术（Flow Cytometry）检测GFP荧光信号。
B. 为C与D部分实验方法的示意图。于第3天，将约2.5×10^5个THP-1细胞（其中约2.5×10^4个已被人巨细胞病毒（Human Cytomegalovirus, HCMV）感染）接种至约2.5×10^5个人真皮成纤维细胞（Human Dermal Fibroblasts, HDFs）的单层培养体系中，随后每3天通过共聚焦成像（Confocal Imaging）监测病毒扩散情况，直至感染后第15天。
C. 展示了TB40/E-GFP与UL88-STOP病毒在感染后第3天和第7天的代表性成像结果。
D. 采用NIS元素软件（NIS element software）以荧光面积为指标量化病毒感染扩散范围，绘图展示了每种病毒的荧光面积倍数变化。
E. 以感染复数0.05的剂量，用TB40/E-mCh或UL88-STOP-mCh病毒感染MRC-5细胞，于感染后第7天（7 dpi）收集细胞并提取总RNA。采用包含84个干扰素刺激基因（Interferon Stimulated Genes, ISGs）的引物组合，通过实时定量聚合酶链反应（quantitative Polymerase Chain Reaction, qPCR）分析抗病毒基因的表达谱。结果以火山图（Volcano Plot）呈现，对比了TB40/E野生型（WT）人巨细胞病毒与UL88-STOP突变株感染后的差异调控基因。
F. 展示了在人巨细胞病毒感染中已知会被调控的干扰素刺激基因，及其在TB40/E野生型与UL88-STOP突变株感染细胞间的mRNA相对倍数变化（以甘油醛-3-磷酸脱氢酶（Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH）作为内参基因进行标准化）。该图表将基因分为三类：TB40/E野生型与UL88-STOP感染组间无显著差异（绿色框）、表达下调（红色框）以及表达上调（蓝色框）。所有实验结果均来自三次生物学重复。