Inhibition of HDACs disrupts DNMT1 association with Hsp90. Inhibition of HDACs disrupts DNMT1 association with Hsp90. A to C. LBH589 increases acetylated lysine residues on Hsp90 and abolishes Hsp90 association with DNMT1. A. MDA-MB-231 cells were treated with 100 nmol/L LBH589 for 12 to 48 h. B and C. MCF-7 and T47D cells were treated with 100 nmol/L LBH589 for 24 h. A and B. Nuclear extracts were prepared and immunoprecipitated by anti-Hsp90 antibody, and immunocomplexes were analyzed by Western blot with anti-acetylated lysine (Ac-K), anti-DNMT1, or anti-Hsp90 antibody. C. DNMT1 protein levels from nuclear extracts were determined by Western blot analysis. A representative Western blot from three independent experiments is shown. D and E. Disruption of DNMT1 association with Hsp90 after HDAC inhibitor treatment can be rescued by proteasome inhibitors. D. MDA-MB-231 cells were treated with 10 μmol/L MG-132 or 100 nmol/L LBH589 for 24 h or the sequence of MG-132 for 6 h followed by exposure to LBH589 for 24 h. E. MDA-MB-435 cells were treated with 3 μmol/L lactacystin (LAC) for 24 h or SAHA for 12 or 24 h or the sequence of lactacystin for 6 h followed by exposure to SAHA for 12 or 24 h. Hsp90 was immunoprecipitated from nuclear extracts, and the blot was probed for Hsp90 and DNMT1. A representative Western blot is shown. Columns, mean densitometric quantification of the DNMT1 protein expression of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, compared with the untreated cells. Qun Zhou et al. Mol Cancer Res 2008;6:873-883 ©2008 by American Association for Cancer Research