Modulation of Notch transcription for subset of retested genes. A. The two constitutively active Notch constructs used to determine epistatic relationships in the pathway, NΔecn and Nicd. NΔecn is a truncated form of N missing the extracellular domain that is initially membrane bound. NΔecn undergoes constitutive cleavage to form the soluble Nicd that is transported to the nucleus to activate transcription. Su(H) is the canonical Notch pathway transcription factor that represses transcription in the absence of Nicd and is essential for the Nicd activated transcription of targets such as E(spl)m3. B. Transcriptional response to RNAi treatment of selected retested genes that promote Notch signaling. The E(spl)m3 reporter was induced with either NΔecn or Nicd or left in the uninduced repressed state. All three classes down regulate both soluble (Nicd) and membrane bound (NΔecn) Notch-induced signal, yet have different effects on the E(spl)m3 promoter in the absence of active Notch. Class I genes have positive, Class II neutral and Class III negative effect on the repressed signal. C. Transcriptional response to RNAi treatment of selected retested genes that repress Notch signaling. Two classes of hits are noted. Class IV is only effective on the membrane bound form of Notch (NΔecn), while class V is effective on both membrane bound and soluble forms (Nicd). Error bars represent the standard error of the mean (SEM). *Significant deviation from control RNAi treatment, calculated by two-tailed t-test with a p-value < 0.05 (Additional file 5 for full statistics).