Figure 1.

H₂O₂ induces accumulation of PML-NBs and deacetylation of PML in HeLa cells. (a) HeLa cells were treated with 4 or 8 mM H₂O₂ for 1 h. Cells were immunostained with an anti-PML antibody followed by fluorescence microscopy (original magnification × 200). (b) Numbers of PML-NBs in each cell were counted. Over 100 cells in duplicate experiments were counted and presented as the mean±S.D. Unpaired two-tail t-tests (**P<0.01 and ***P<0.001) were used for statistical analyses. (c) HeLa cells were treated with the indicated concentration of H₂O₂ for 1 h. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting with the indicated antibodies. (d and e) HeLa cells (d) or the cells transfected with HA-tagged PML4 (e) were treated with 0, 4, or 8 mM of H₂O₂ for 0.5 h. Whole-cell extracts (WCEs) were prepared and immunoprecipitated with anti-PML (d) or anti-HA (e) antibodies followed by immunoblotting with anti-acetyl-lysine and anti-PML(d) or anti-HA (e) antibodies. (f) HeLa cells were transfected with HA-PML4 and FLAG-SIRTs. WCEs were analyzed by immunoprecipitation with anti-HA antibodies followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies. The expression of SIRTs was examined by immunoblotting with anti-FLAG antibodies. The composite image in (f) was spliced from a western blot