Fig. 3

Vpr expression induces Topo1 stress. a Reduced expression of Topo1 under Vpr expression. Mit-23 cells were treated with Dox (3 μg/ml, 2 days) or CPT (20 μM, 1 h). By WB analysis, Topo1 and Ku70 as an internal control were detected. Relative intensities of Topo1 were calculated by normalizing the amounts of Topo1 by Ku70 (Left panel). Immunohistochemical analysis detected Topo1 and DNA (Hoechst-33258) (Right panel). ΔVpr cells were used as a control cell line without exogenous Vpr. b Formation of Topo1-cc under Vpr expression. RADAR analysis was performed on Mit-23 and ΔVpr cells, which were treated with Dox or CPT in the presence or absence of MG-132 (50 μM, 2 h). c Ubiquitination of Topo1 under Vpr expression. Mit-23 cells transfected with 3×FLAG/6×His-Ubiquitin were treated with Dox or CPT in the presence of MG-132, and immunoprecipitated with α-Topo1 antibody. Relative intensities of ubiquitinated Topo1 in the immunoprecipitates are shown. d Vpr induces SUMOylation of Topo1. Immunoprecipitation using α-Topo1 was performed in Mit-23 cells transfected with 3×FLAG/6×His-SUMO-1. CPT and Vpr expression induced modification of Topo1 with SUMO-1. Relative intensities of SUMOylated Topo1 in the immunoprecipitates are shown