USP37 and HBx act in synergy to deregulate cell Cycle.

(A) Huh7 cells were transfected with Vector, Flag-USP37 and Flag-USP37-DD construct and the levels of USP37 protein were measured by western blotting (WB). (B) Ubiquitination assay was performed with lysates from cells transiently expressing Vector, HA-HBx, Flag-USP37, Flag-USP37-DD and Myc-Ubi as indicated (Cells in 100 mm dish were transfected with 4 µg Vector and 2 µg Myc-ubiquitin; 2 µg Vector, 2 µg HA-HBx and 2 µg Myc-ubiquitin; 2 µg Flag-USP37, 2 µg HA-HBx and 2 µg Myc-ubiquitin or 2 µg Flag-USP37-DD, 2 µg HA-HBx and 2 µg Myc-ubiquitin) and treated with 20 µM MG132 for 6 h, by immunoprecipitating Cyclin A. Immuno-complexes were eluted and western blotted with α-Ubiquitin antibody. (C) Cyclin A expression was chased in IHH cells transiently transfected with Vector, HA-HBx, Flag-USP37 and Flag-USP37-DD as indicated and harvested at indicated time intervals post 72 hrs serum starvation. (D) IHH cells transfected with Vector control, HA-HBx, Flag-USP37 and Flag-USP37-DD as indicated, were synchronized in G0/G1 phase by Serum starvation followed by harvesting at indicated time points. Cells in different phases of cell cycle were analyzed by flow cytometry. Values are represented as bar diagrams (E) Brd-U incorporation assay was carried out in Huh7 cells transfected with Vector control, HA-HBx, Flag-USP37 and Flag-USP37-DD as indicated, by incorporating BrdU followed by staining with antibody against BrdU and counterstaining with DAPI to observe actively replicating cells as seen in the representative confocal images. Scale bar represents 50 µm.(A) 将Huh7细胞分别转染空载体（Vector）、Flag-USP37及Flag-USP37-DD重组质粒，采用蛋白质印迹法（western blotting，WB）检测USP37蛋白的表达水平。 (B) 泛素化检测实验采用如下方式进行：将分别转染空载体、HA-HBx、Flag-USP37、Flag-USP37-DD及Myc-Ubi的细胞裂解液用于实验（具体转染体系：100mm培养皿中，转染4μg空载体与2μg Myc-泛素；或2μg空载体、2μg HA-HBx与2μg Myc-泛素；或2μg Flag-USP37、2μg HA-HBx与2μg Myc-泛素；或2μg Flag-USP37-DD、2μg HA-HBx与2μg Myc-泛素），并以20μM MG132处理细胞6小时，随后针对细胞周期蛋白A（Cyclin A）进行免疫沉淀（immunoprecipitation）。将免疫复合物洗脱后，使用泛素抗体进行蛋白质印迹检测。 (C) 对经血清饥饿72小时后，分别转染空载体、HA-HBx、Flag-USP37及Flag-USP37-DD的IHH细胞进行追踪实验，按指定时间间隔收取细胞样本，检测细胞周期蛋白A（Cyclin A）的表达水平。 (D) 将分别转染空载体对照、HA-HBx、Flag-USP37及Flag-USP37-DD的IHH细胞通过血清饥饿法同步化至G0/G1期，随后按指定时间点收取细胞。通过流式细胞术分析细胞周期各时相的分布情况，结果以柱状图呈现。 (E) 对分别转染空载体对照、HA-HBx、Flag-USP37及Flag-USP37-DD的Huh7细胞进行BrdU掺入实验：向培养基中加入BrdU进行孵育，随后使用BrdU抗体进行免疫荧光染色，并用4',6-二脒基-2-苯基吲哚（DAPI）复染，以观察活跃增殖的细胞，结果如代表性共聚焦显微镜图像所示。比例尺为50μm。