Figure 10.

Inhibition of nuclear factor of activated T-cells 1-interaction with the c-MYC promoter by cardiac glycosides. (a) Cardiac glycoside effects on nuclear factor of activated T-cells (NFAT) 1 binding to the c-MYC promoter. Cells were treated with vehicle, digitoxin (500 nM), or oleandrin (100 nM) for 6 h. Chromatin immunoprecipitation (ChIP) was performed with either NFAT1 antibody or IgG (Immunoglobulin G) control. Quantitative PCR (polymerase chain reaction) analysis was used to estimate the level of MYC promoter or upstream control chromatin isolated during ChIP analysis. Values were normalized to those found during qPCR amplification of material from 10% of input sheared chromatin (*P < 0.05). (b) Schematic representation of qPCR amplicons for ChIP analysis of human c-Myc promoter element, including location of the TIE (TGFβ interacting element) for nuclear factor of activated T-cells (NFAT) binding. NFAT1 association was assayed by amplification of a nearby 67 bp product at position-59 from the transcription start site. The control ChIP background pull-down was assayed by amplification of a distant 110 bp product at position-2614 from the transcription start site