Figure 3. Doxo induces H2AX eviction and attenuates DDR.

(a) Part of the nucleus of MelJuSo cells expressing PAGFP-H2AX was activated before exposure to Doxo. The boundaries of nuclei are indicated. Fluorescence intensities are shown in false colours. Scale bar, 10 μm. (b) MelJuSo cells were treated with 9 μM Doxo or 60 μM Etop for 2 h before fixation and stained for γ-H2AX (top panel in red). Bottom panel in blue indicates DAPI staining of the nuclei of cells. C, untreated control. Scale bar, 10 μm. (c) MelJuSo cells were treated with 9 μM Doxo or 60 μM Etop and lysed at indicated time points before analyses of γ-H2AX by SDS–polyacrylamide gel electrophoresis (PAGE) and western blotting (WB). Tubulin is used as a loading control and the positions of molecular weight markers are indicated. (d) MelJuSo cells were exposed to various concentrations of Doxo, Etop or Acla for 2 h. C, untreated control. Drugs were removed by extensive washing. DNA double-strand breaks, immediately after 2 h drug treatment or 8 h post drug removal were quantified by constant-field gel electrophoresis and expressed as percentage of total DNA (n=3 independent experiments, error bar indicates s.d.). Western blotting indicates the γ-H2AX response after 2 h drug treatment at different concentrations; tubulin is shown as loading control. (e) MelJuSo cells were exposed to 9 μM Doxo, 60 μM Etop or 20 μM Acla for 2 h. Drugs were removed and further cultured for the times indicated. Cells were lysed, separated by SDS–PAGE and WB was probed with the antibodies indicated. Actin is used as loading control and positions of marker are indicated. C, untreated control.