Figure 5

SIRT3 was expelled from the nucleus to the mitochondria by Oroxylin A upon cellular oxidative stress. (a) MDA-MB-231 cells were treated with Oroxylin A for 48 h. Mitochondrial and nuclear fractions were isolated after treatment and subjected to western blot analysis for full-length band and cleaved band of SIRT3. (b) MDA-MB-231 and MCF-7 cells were treated with Oroxylin A upon different concentrations or time. Quantification of ROS was detected using fluorescent dye DCFH-DA by FACSCalibur flow cytometry at Ex/Em of 488 nm/525 nm. Bars, S.D., *P<0.05 or **P<0.01 versus Oroxylin A-treated without siRNA group of MDA-MB-231 cells, ^#P<0.05 or ^##P<0.01 versus without siRNA group of MCF-7 cells. (c) Cells were treated with 1 mM H₂O₂ for 36 h or 200 Oroxylin A for 48 h, respectively. Immunofluorescence experiment performed in MDA-MB-231 and MCF-7 cells upon oxidative stress or Oroxylin A treatment using antibodies specific to full-length of and cleaved SIRT3, DAPI and Mitotracker. (d) MDA-MB-231 cells were pretreated with10 mM NAC for 1 h, then treated with 1 mM H₂O₂ for 36 h or 200 μ M Oroxylin A for 48 h, respectively. Mitochondrial fractions were isolated after treatment and subjected to western blot analysis for HK II and SIRT3