Cell cycle– and proliferation-associated genes differentially expressed by Notch1-IC, Notch2-IC, and EBNA2. Cells were treated as outlined in Figure 1B and harvested at the indicated time points. mRNA levels were quantified by qRT-PCR. mRNA levels were normalized to the expression of ribosomal protein genes and standardized to the value at the time of induction (0 hours). Values represent fold induction of mRNA levels compared with the mRNA levels at 0 hours, which was set to 1. Values are representative for 3 independent qRT-PCRs of at least 2 biologic replicates. (A) Cell cycle–associated genes, which were up-regulated late by EBNA2. These genes were in all cases up-regulated stronger by EBNA2 than by Notch1/2-IC. Cyclins (CCN); cyclin-dependent kinases (CDK). (B) CCN and CDK, which were up-regulated early by EBNA2 and were regulated similarly by Notch1/2-IC. (C) qRT-PCR analysis of the proliferation-associated genes phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and MYC, which were up-regulated early and strongly by EBNA2, and only marginally by Notch1/2-IC. (D) Protein expression of MYC and PIK3R1 by Notch1/2-IC and EBNA2: Notch1-IC/EREB, Notch2-IC/EREB, and CAT/EREB cells were deprived of estrogen for 3 days. Whole-cell lysates were harvested from growth-arrested cells (0 hours), from cells that were stimulated with doxycycline for 8 hours or 24 hours (Notch1-IC, Notch2-IC, CAT) or from CAT/EREB cells, which were stimulated with estrogen for 4 hours or 24 hours (EBNA2). In addition, whole-cell extracts were prepared from CAT/EREB cells continuously growing in the presence of estrogen (pr). Membranes were stained with antibodies raised against human Myc protein and PIK3R1 (recognizing p85α, p55α, and p50α). Equal protein loading was controlled by staining with an α-tubulin antibody. Error bars in panels A through C represent SDs.