Figure 8C, D

<b>A. </b>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>B.</b> Schematic representation of the experiment methodology for <b>C</b> and <b>D</b>.<b> </b>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. <b>C</b>. Representative images of TB40/E-GFP and UL88-STOP from day 3 and day 7 are shown. <b>D.</b> The spread of infection was quantified as the area of fluorescence using the NIS element software.<b> </b>Graph shows fold change in area of fluorescence for each virus. <b>E. </b>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. <b>F</b>. 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. THP-1细胞以感染复数（multiplicity of infection, MOI）20，经离心感染法分别感染无荧光的TB40/E、UL88-STOP及UL88-Rev病毒；后两种病毒携带由IE2启动子驱动的绿色荧光蛋白（green fluorescent protein, GFP）表达盒，同时设置未感染对照组。感染后第5天（d5）通过流式细胞术检测GFP荧光信号。
B. 本部分为对应后续C、D实验的实验方法示意图。实验第3天，将约2.5×10^5个THP-1细胞（其中约2.5×10^4个已被人类巨细胞病毒（human cytomegalovirus, HCMV）感染）接种至约2.5×10^5个人包皮成纤维细胞（human dermal fibroblasts, HDFs）的单层培养板中；随后每3天通过共聚焦成像监测病毒扩散情况，直至感染后第15天（d15）。
C. 展示了TB40/E-GFP与UL88-STOP病毒在感染后第3天及第7天的代表性成像结果。
D. 采用NIS Elements软件通过荧光面积定量感染扩散程度，所得图表展示各病毒组的荧光面积倍数变化。
E. 以感染复数0.05分别感染MRC-5细胞，所用病毒株为TB40/E-mCh或UL88-STOP-mCh。感染后第7天（7 dpi）收集细胞并提取总RNA，通过针对84种干扰素刺激基因（interferon-stimulated genes, ISGs）的qPCR芯片检测抗病毒基因的表达谱。结果以火山图形式呈现，对比TB40/E野生型（WT）HCMV与UL88-STOP HCMV感染组的差异调控基因。
F. 展示了已知在HCMV感染中发生表达调控的干扰素刺激基因（ISGs），其mRNA水平在TB40/E WT与UL88-STOP病毒感染组间的相对倍数变化（以GAPDH作为内参基因进行标准化）。该图表将基因分为三类：TB40/E WT与UL88-STOP感染组间无显著差异（绿色框）、表达下调（红色框）及表达上调（蓝色框）。所有实验结果均来自三次生物学重复。