Testing for Ingredients that Inhibit MUC1 CD Dimerization. The 72 amino-acid MUC1 C cytoplasmic domain contains cysteine residues at positions 1 and 3 which can be essential for its dimerization. To build up an analysis for pinpointing inhibitors of MUC1 CD dimerization, 96 HDAC1 inhibitor well plates were first coated with purified MUC1 CD. Biotinylated MUC1 CD was then added to the wells, and its interaction with bound MUC1 CD was detected with streptavidin HRP. Quantitation of the signals was established with EnVision. Using this technique, six libraries containing significantly more than 5000 compounds were tested for molecules that block the formation of MUC1 CD dimers. Original screens were done in the presence of substances in a concentration of 100 _M. Ingredients that inhibited dimerization by more than 50% were chosen for further evaluation. Using these criteria, the proportion of good compounds ranged from number 1 to almost 4% with regards to the library. Identification of Apigenin as an Inhibitor of MUC1 Disc Dimerization. In line with the testing, we discovered the flavone apigenin Metastasis as one candidate inhibitor. In contrast to car, 100 _M apigenin inhibited MUC1 CD dimerization by approximately 80%. In comparison, the structurally relevant flavone baicalein had little, if any, impact. Investigation of apigenin over a variety of levels further demonstrated 5000-mile inhibition of MUC1 CD dimerization at 76 _M. To give these findings, reports of MUC1 CD dimerization were conducted using soluble unbound protein. Previous work showed that the 10 kDa MUC1 CD Fig. 4. Apigenin inhibits MUC1 expression in MCF 7 cells. A, MCF 7 cells were treated with DMSO, 75 _M apigenin, or 75 _M baicalein for 3 days. Total RNA was assayed for MUC1 mRNA amounts by quantitative reverse transcription polymerase chain reaction. The are expressed as relative MUC1 mRNA amounts compared with that obtained in cells treated with DMSO. C and B, MCF 7 cells were treated with the 75 _M or Decitabine price the indicated concentrations of baicalein and apigenin for 3 days. Nuclear and total cell lysates were immunoblotted with the indicated antibodies. D, lysates from MCF 7 cells attacked to stably express a get a handle on lentivirus and one with a MUC1 small hairpin RNA were immunoblotted with the indicated antibodies. E, the suggested MCF 7 cells were treated with 75 _M apigenin for 3 days. Viable cellular number was determined by the MTS assay. The are expressed as the percentage of control cellular number in the presence of DMSO. monomer forms 20 kDa dimers in solution. The synthesis of MUC1 CD dimers was completely blocked by apigenin, although baicalein had little effect, as detected by immunoblot analysis. Transfection of cells with GFP MUC1 CD and Flag MUC1 CD has also been used to gauge the development of MUC1 CD dimers in coimmunoprecipitation assays. In this regard, immunoblot analysis of anti Flag precipitates with anti GFP quickly detected MUC1 CD dimerization in the absence of treatment.