Figure 6.

Deacetylation of PML is required for H₂O₂-induced accumulation of PML-NBs. (a) H₂O₂ induces SIRT1 nuclear accumulation of SIRT1. HeLa cells were treated with or without 4 or 8 mM of H₂O₂ for 0.5 h. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting. (b) HeLa cells stably expressing shCtrl and shSIRT1 were treated with H₂O₂ for 1 h. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting. SE, shorter exposure; LE, longer exposure. (c) HeLa cells expressing shCtrl and shSIRT1 stably were treated with H₂O₂ for 1 h followed by immunofluorescence microscopy probed with anti-PML (original magnification × 200). (d) HeLa cells stably expressing the indicated shRNAs were transfected with shRNA-resistant SIRT1. Nuclear fractions were prepared and subjected to western blotting. (e) HeLa cells stably expressing the indicated shRNAs were treated with H₂O₂ for 1 h. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting. (f) HeLa cells expressing shCtrl or shSIRT1 stably were transfected with HA-PML4 and Myc-SIRT5 (wild-type or H158Y mutant). Whole cell extracts (WCEs) were prepared and immunoprecipitated with anti-HA antibody. WCEs and immunopellets were subjected to immunoblotting with anti-Myc, anti-acetyl-lysine, and anti-HA antibodies. (g) HeLa cells stably expressing the indicated SIRT1 shRNA were transfected with wild-type or mutant SIRT5. Nuclear fractions were prepared and analyzed as in (d). (h) HeLa cells stably expressing shCtrl or shSIRT1 were transfected with Myc-SIRT5 (wild-type or H158Y mutant). Cells were immunostained with anti-PML and anti-Myc antibodies and images were taken by fluorescence microscope