miR-34a directly regulates MYB expression. (A) miR-34a regulates luciferase activity of a reporter vector containing either the full-length 3′-UTR of MYB (left) or individual predicted MRE (right). Relative luciferase activity was assayed in 293T cells 48 hours after cotransfection with a luciferase reporter plasmid and the indicated miRNA mimic. AS-34a designates a reverse complementary sequence of miR-34a. (B) Overexpression of miR-34a in K562 cells down-regulates MYB protein and mRNA. K562 cells, transfected with the indicated miRNA mimic, were analyzed for MYB protein (left) and mRNA (right) by immunoblot or quantitative PCR, respectively, 48 hours later. Protein expression relative to α-tubulin was quantified by densitometry. mRNA normalized to GAPDH mRNA is shown relative to the ratio in K562 cells transfected with a miRNA mimic control. (C) MYB knockdown (right) up-regulates the MK-specific marker CD41 in cells treated with a suboptimal amount of TPA (0.1 nM). As positive control for CD41 induction, K562 cells were treated with 1 nM TPA.