Skip to main content
. 2008 Nov 28;283(48):33110–33118. doi: 10.1074/jbc.M804255200

FIGURE 3.

FIGURE 3.

BTG2/TIS21 up-regulated generation of ROS induced by doxorubicin treatment. A, fluorescence-activated cell sorter analysis showing DOXO-induced ROS in HeLa cells. The cells were treated with DOXO (1.0 μg/ml) for 12 h, and DCFDA fluorescence generated by ROS was measured by flow cytometry. B, Western blot analysis showing the inhibition of DOXO-induced cell death by pretreatment with NAC. HeLa cells were pretreated for 1 h with or without indicated concentrations of NAC and subsequently treated with DOXO (1.0 μg/ml) for 24 h. Note the significant decrease of lamin A/C cleavage by NAC. C, inhibition of pyrogallol-induced cell death by NAC. Upper panel, HeLa cells were incubated with indicated concentrations of PYRO, superoxide generator, with NBT for 3 h, and NBT reduced by superoxide anion was then examined by measuring the A575. ⋆, p < 0.05 versus PYRO (0 μm); #, p < 0.05 versus PYRO (50 μm). Lower panel, HeLa cells were pretreated with either PYRO (50 μm) or NAC (5 mm) for 48 h, and then cleavage of PARP was examined by Western blot analysis. Note the significant inhibition of PYRO-induced cell death by NAC. D, TIS21 enhanced PYRO-induced cell death, whereas NAC inhibited cell death. HeLa cells were infected with 50 m.o.i. of Ad-TIS21 or Ad-β-Gal as a control, and then cell death was induced by treatment of the cells with PYRO (50 μm) for 48 h with or without NAC (5 mm) pretreatment. Upper panel, Western blotting to determine the cleavage of PARP. Lower panel, viable cells were determined by trypan blue staining. The data indicate inhibition of PYRO-induced changes by NAC, suggesting in vivo conversion of PYRO-induced superoxide to H2O2. ⋆, p < 0.05 versus PYRO-treated, Ad-β-Gal-infected; #, p < 0.05 versus PYRO-treated Ad-TIS21-infected. E, inhibition of DOXO-induced ROS generation by sh-BTG2 RNA. HeLa cells infected with shRNA, H4 and H5, virus, or empty vector (Con) were exposed to 1.0 μg/ml of DOXO for 12 h, and DCFDA fluorescence produced by ROS was measured by flow cytometry. ⋆, p < 0.05 versus Con with DOXO treatment. F, enhancement of DOXO-induced ROS generation by Ad-TIS21 infection and efficient inhibition by pretreatment with NAC. HeLa cells were infected with either 50 m.o.i. of Ad-TIS21 or Ad-β-Gal as a control, and then cell death was induced by treatment with either DMSO or DOXO (1.0 μg/ml) for 12 h with or without NAC (5 mm) pretreatment. DCFDA fluorescence produced by ROS was measured by flow cytometry. ⋆, p < 0.05 versus DOXO-treated and Ad-β-Gal infected cells. The data represent results of a typical experiment or average values with standard deviations from four independent experiments.