Fig 6. Formation of salt-stable closed-clamp intermediates induced by ICRF-193 treatment.

(A) HEK293 cells in a 35-mm glass-bottom dish were transfected with the FLAG-topo IIβ-EGFP expression vector. The live cells were imaged using a 40× objective lens. ICRF-193 (7 μM) was added for 15 min. Scale bar = 5 μm. (B) The clamping assay was performed as described in materials and methods. FLAG and EGFP-tagged proteins were detected by using the anti-FLAG tag antibody. (C) Relative band intensities in (B) were calculated from band densitometry using Image J software. The experiments were performed in triplicate, and the results are indicated as mean ± S.D. Circles represent individual data points. Asterisks (P < 0.001) indicate a significant deviation from WT (Student’s t-test).