CDK inhibitors affect c-Myb activity. (A) Reporter-gene assays. A Myb-responsive reporter plasmid was transfected into QT6 cells along with plasmids expressing A-Myb, B-Myb, or c-Myb with or without a plasmid expressing p27 Kip1. Data are plotted as mean fold stimulation relative to the samples lacking p27. Error bars illustrate the range of replicate assays performed in parallel. (B) Endogenous gene activation assay. QT6 cells were transfected with combinations of plasmids expressing NF-M plus c-Myb and various CDK inhibitors as indicated at the top. After 2 days, cells were harvested and analyzed by Northern blotting using a probe specific for Mim1 then stripped and sequentially rehybridized with probes for lysozyme and beta-actin (as an RNA loading control). Western blotting showed that equivalent levels of c-Myb were expressed in all the samples (not shown). (C) Effects of cell-cycle regulators. HD-11 cells were transfected with plasmids expressing c-Myb alone (lane 1) or together with plasmids expressing the indicated combinations of cell-cycle regulators. After 2 days, RNA was harvested and analyzed by Northern blotting for the expression of the endogenous Mim1 or beta-actin genes. (D) Regulation summary. The data suggest a regulatory pathway in which increased cyclin D1/CDK inhibits the ability of c-Myb to activate specific target genes. Expression of CDK inhibitors p16, p21, or p27 stimulates c-Myb by inhibiting the activity of the cyclin D1/CDK complex.