Bortezomib treatment down-regulates DNMT1 expression in vitro and in vivo. (A) Bortezomib decreased DNMT1 mRNA transcription (left) and protein expression (right) in a dose-dependent manner. (B) Bortezomib decreased DNMT1 mRNA (left) and protein (right) in a time-dependent fashion. MV4-11 cells were incubated with different concentration of bortezomib (0, 1, 6, 20, 60, and 100 nM) for 24 hours or 60 nM bortezomib for indicated time periods. DNMT1 mRNA levels were examined by quantitative real-time RT-PCR (A,B left panel), and DNMT1 protein levels were detected by Western blot (A,B right panel). (C) Down-regulation of DNMT1 protein expression was observed in AML patient primary blasts treated ex vivo with bortezomib. Primary cells were exposed to 20 nM or 60 nM bortezomib for 48 hours, and the cell lysates were applied to Western blot for DNMT1 protein (left panel). Quantification of DNMT1 protein level normalized by β-actin is shown (right panel). (D,E) DNMT1 protein expression was repressed in tumors from MV4-11 (D) and K562 (E) xenograft mice following treatment with a single dose (2 mg/kg) of bortezomib (intravenous bolus). Error bars represent SD.