Figure 2. THZ1 delays the ubiquitin-dependent degradation of topotecan-induced TOP1-DPC in DMS114 SCLC cells.

A. Cells treated with TPT (1 μM) for the indicated times in the presence and absence of THZ1 (1 μM) were collected for ICE assay to detect TOP1-DPC with anti-TOP1 antibody. THZ1 (1 μM) was added 2 h prior to the TPT treatment. Total DNA (dsDNA, right panel) was detected using anti-dsDNA antibody and served as loading control.

B. Densitometric analyses comparing the relative integrated densities of TOP1-DPCs from Independent experiments as shown in panel A. *: p < 0.05

C. Scheme for the Detection of Ubiquitylated and SUMOylated TOP-DPCs (DUST) assay.

D.Cells were pretreated with THZ1 (1 μM) or BTZ (1 μM) for 2 hours prior to cotreatment with TPT (20 μM) for 30 min and were subjected to DUST assay (upper panel) and total TOP1-DPCs (middle panel) using anti-ubiquitin and anti-TOP1 antibodies, respectively.

E. Quantitation of ubiquitylated TOP1-DPCs and total TOP1-DPCs. Upper panel: Densitometric analyses comparing relative integrated densities of Ub signals from independent experiments as shown in panel D. Lower panel: Densitometric analyses comparing relative integrated densities of TOP1-DPC signals from independent experiments as shown in D.

F. Cells were pretreated with THZ1 (1 μM) or BTZ for 2 hours prior to cotreatment with TPT (20 μM) for 1 or 4 hours. Cell were then lysed and digested with benzonase for Western blotting for immunodetection of TOP1 using anti-TOP1 antibody.

G. Cells were treated with TPT (1 μM) in the presence and absence of THZ1 (1 μM) and collected for immunofluorescence for detection of γH2AX.