Bortezomib induces re-expression of methylation-silenced genes via promoter hypomethylation. (A) Bortezomib increased expression of RIL gene reportedly to be methylated and down-regulated in HCT116 and ML-1 cells. HCT116 and ML-1 cells were treated with bortezomib for indicated time points and dosage; RIL gene expression was measured by real-time RT-PCR. Error bars represent SD. (B) Bortezomib induced DNMT1 down-regulation in HCT116 and ML-1 cells. HCT116 and ML-1 cells were treated with bortezomib, and Western blot was performed using antibodies against DNMT1 or HDAC1 (control). (C) Hypomethylation of RIL promoter by bortezomib treatment in ML-1 cells. DNA (1 μg) from bortezomib-treated or untreated ML-1 cells were digested by HpaII or BstuI, and PCR was performed using primers specific for RIL gene promoter. HpaII indicates no digestion, then hypermethylated; BstuI, no digestion, then hypomethylated.