Aurka is a direct Myc target gene, whereas Aurkb is indirectly up-regulated by Myc. (A) Top panel: Immunoblot analysis of NIH-3T3 cells infected with Myc or GFP control retrovirus. Vertical lines have been inserted to indicate a repositioned gel. Bottom panel: NIH-3T3 cells were transiently transfected with the indicated firefly luciferase promoter-reporter constructs and assessed for relative luciferase activity. Shown is the relative luciferase activity in Myc-expressing versus GFP-only expressing cells. Renilla luciferase plasmids were cotransfected to normalize for transfection efficiency. Bars represent the mean ± SEM of 3 independent experiments, each performed in duplicate. *P < .05. (B) Right panel: Immunoblot analysis of NIH-3T3 cells infected with retroviruses encoding MSCV-Myc-ER-IRES-Puro. Left panel: After puromycin selection, cells expressing Myc-ER were cultured in the presence or absence of 4HT and/or cycloheximide (Chx) for 4 hours to activate preexisting Myc-ER in the presence or absence of protein synthesis. Ethanol (EtOH) was the vehicle for 4HT. The cells were harvested and RNA prepared for quantitative RT-PCR in which primers directed against Aurka, Aurkb, Ldha, and Ubiquitin (Ub) were used. Levels of mRNA were standardized to the expression of Ub. Bars represent the mean ± SD from 3 experiments. The up-regulation of all genes is significant on Myc activation in the absence of Chx (P < .05). The induction of Aurka by Myc is significant in the presence of Chx. *P < .05. n.s. indicates not significant. (C) ChIP analysis for Myc binding to mouse Aurk genes in Balb/c-3T3 fibroblasts expressing Myc-ER. Top panel: Map of the murine Aurka and Aurkb gene regions analyzed and the location of the E-boxes and amplicons used to detect Myc binding by quantitative PCR after ChIP. Amplicons A1.1, A1.2, A1.3, A2.2, A.2.1, A3.2, and A control detect Aurka, whereas amplicons B1.1, B1.2, B1.3, B2.1, B2.2, and B control detect Aurkb genomic DNA. Chromatin was isolated from cells 6 hours after activation of Myc-ER (+4HT) or uninduced (−4HT) and immunoprecipitated with antibody against c-Myc. Quantitative PCR was carried out on immunoprecipitated chromatin and input chromatin and expressed as percentage total using the calculation % total = 2^{Ct input − Ct ChIP} × % input used for ChIP.²⁸  Shown is the mean of 3 experiments ± SD. *P values comparing −4HT and +4HT samples.