Figure 2A

A-C. Fibroblasts (MRC-5 or HDF) and ARPE-19 were transduced with lentivirus expressing UL88 or vector and subjected to puromycin selection. Cells were harvested for either WB or RNA analysis. A. MRC-5 cells expressing either vector or UL88 were analysed by western blot and probed for MyD88, UL88, and tubulin. Graph shows MyD88 protein levels quantified by densitometry analysis from 4 independent experiments. Results are shown as mean ± SD. *p&lt;0.05. B. Cell lysates from HDFs expressing GFP vector, GFP-UL88, vector, or UL88, were analysed by western blot and probed for MyD88 and tubulin. C. Cell lysates from ARPE-19 cells expressing GFP vector or GFP-UL88 were analysed by western blot and probed for GFP, UL88, MyD88, and tubulin. D. Cells expressing either GFP-vector or GFP-UL88 were stimulated with IL-1β for 6 hours. Cells were fixed in PFA and stained for p65-NF-KB (red), GFP (green), and DAPI (blue). Cells were imaged on the confocal. E. At least ten different fields were images for each sample. The number of nuclei and NF-KB positive nuclei were counted. The percentage of NF-KB positive nuclei is graphed.A~C：将成纤维细胞（MRC-5或人真皮成纤维细胞HDF）与ARPE-19细胞使用携带UL88编码序列的慢病毒（lentivirus）或空载体进行转导，随后以嘌呤霉素（puromycin）进行筛选。收集细胞用于蛋白质免疫印迹（Western Blot，WB）或RNA水平分析。 A. 对表达空载体或UL88的MRC-5细胞开展蛋白质免疫印迹检测，以MyD88、UL88及微管蛋白（tubulin）为靶标进行探针杂交。定量统计图展示了4次独立实验通过光密度扫描分析得到的MyD88蛋白相对表达水平，结果以平均值±标准差（mean ± SD）呈现，*p<0.05。 B. 对分别转染绿色荧光蛋白（Green Fluorescent Protein，GFP）空载体、GFP-UL88融合载体、空载体或UL88编码载体的人真皮成纤维细胞（HDF）的细胞裂解液进行蛋白质免疫印迹分析，以MyD88与微管蛋白为靶标进行探针检测。 C. 对转染GFP空载体或GFP-UL88融合载体的ARPE-19细胞的细胞裂解液开展蛋白质免疫印迹检测，以GFP、UL88、MyD88及微管蛋白为靶标进行探针杂交。 D. 对转染GFP空载体或GFP-UL88融合载体的细胞用白细胞介素1β（Interleukin-1β，IL-1β）刺激6小时，随后用多聚甲醛（Paraformaldehyde，PFA）固定细胞，对p65-核因子κB（p65-NF-κB，红色荧光）、GFP（绿色荧光）及4',6-二脒基-2-苯基吲哚（DAPI，蓝色荧光）进行免疫荧光染色。通过共聚焦显微镜（confocal）采集细胞成像。 E. 每个样本至少采集10个不同视野的图像，计数细胞核总数与NF-κB阳性细胞核数目，绘制NF-κB阳性细胞核占比的统计图表。