Mad1 is a transcriptional target of the granulocyte-restricted, retinoid target gene C/EBPϵ. (A) Induction of Mad1 RNA is delayed following differentiation induction of wild-type MPRO cells with the RARα agonist AGN195183. Wild-type MPROs were treated for the indicated number of days with AGN195183 and Mad1 RNA levels were assessed by Northern blotting as described. (B) Expression of Mad1 protein is also delayed following treatment with AGN195183. Mad1 protein levels were analyzed using laser-assisted confocal microscopy at the indicated time points. (C) Induction of C/EBPϵ by the RARα agonist AGN195183 occurs rapidly and precedes the induction of Mad1. Quantitative real-time PCR was performed to determine the expression of C/EBPϵ and Mad1 following treatment of MPROs with AGN195183 for the indicated time periods. Results are normalized to the expression of β₂-microglobulin. Error bars represent SEM from 3 PCR reactions. (D) Mad1 is a transcriptional target of C/EBPϵ. Wild-type C/EBPϵ-ER MPROs were treated with tamoxifen, tamoxifen and CHX, or CHX alone for the indicated time points; then RNA was prepared and analyzed for Mad1 expression. (E) C/EBPϵ-ER or pBabe MPROs were treated for 0, 14, and 24 hours with tamoxifen, fixed, and then expression of Mad1 protein analyzed using laser-assisted confocal microscopy.