Figure 4

The effect of N-terminal TOP2B mutants on the DNA-protective effect of DRZ. (A) and (B) HTETOP cells were transfected with siRNA oligonucleotides targeting 3-UTR region of TOP2B mRNA for 48 h. Cells shown in (B) were transfected 24 h earlier with plasmids expressing YFP-tagged wild type TOP2B. Both endogenous and YFP-tagged TOP2B protein levels were detected by antibody specific for human TOP2B. (C) HTETOP cells were transfected with plasmid expressing WT or mutated YFP-tagged TOP2B. Twenty-four hours later 1 μg/ml TET and 50 nM TOP2B 3-UTR siRNA were added for another 24 h, finally the cells were exposed to 1 μM DOX, 100 μM DRZ alone or in combination (DRZ was given 30 min before DOX) for further 24 h. γ-H2AX accumulation and TOP2A protein level were determined by Western blot. Φ: untreated cells, as the positive control for TOP2A detection.