Fig. 2: Nanog inhibits Rad51-mediated γH2AX removal in human cancer cells.

a Nanog enhanced level of γH2AX. The plasmid expressing GFP-tagged Nanog was transfected in HeLa cells for 48 h. Mock vector was used as control. Whole cell proteins and histones were extracted and subjected to SDS-PAGE, respectively. b, c Nanog overexpression increased γH2AX foci. HeLa cells overexpressing GFP-tagged Nanog were stained with the anti-γH2AX antibody (red) and DAPI (blue). The nuclei containing more than 10 foci were considered to be positive. d Nanog-promoted γH2AX activation. 293 cells were transfected with different amounts of plasmid expressing GFP-tagged Nanog (+: 2 μg; ++: 4 μg; +++: 6 μg; ++++: 8 μg). Whole cell proteins and histones were extracted and subjected to SDS-PAGE, respectively. e The blot band intensities from d were quantitated by MultiGauge software (Fujifilm). The data was normalized to loading controls with the antibody against H3. f HeLa cells ectopically expressing Nanog were transfected with different amounts of plasmid expressing HA-tagged Rad51 (+: 0.1 μg; ++: 0.5 μg; +++: 1 μg). Whole cell proteins and histones were extracted and subjected to SDS-PAGE, respectively. g The blot band intensities of f were quantitated by MultiGauge software (Fujifilm). The data was normalized to loading controls with the antibody against H3. h, i HeLa cells ectopically expressing Rad51 were transfected with different amounts of plasmid expressing HA-tagged Nanog (+: 1 μg; ++: 2 μg; +++: 4 μg). Similar strategies were performed with f and g. The data are based on three independent repeats, and presented as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05 (Student’s t-test).