Right panel: CD47 protein levels are lower on cells in tumours generated from MCF7 cells with NFKB1 knockdown (red) versus those without knockdown (controls with no shRNA (turquoise) or with control shRNA (orange)). Left panel: TurboRFP (reporter) expression levels are similar in cells treated with turboRFP-control shRNA (yellow) versus turboRFP-NFKB1 shRNA (pink). Lower panels: flow cytometry analysis of cells from dissociated tumours that had grown for 6 weeks. NFKB1 knockdown led to smaller tumour sizes. ( b) Upper panels: Images of tumours 6 weeks after injection of MCF7 cells that were: untreated (control left) infected with turboRFP-control shRNA (middle) or infected with turboRFP-NFKB1 shRNA (right). Black dots: Control shRNA treated breast tumours derived from MCF7-Luc in xenotransplants. Student's unpaired t-test for independent samples was performed. ( a) NFKB1 shRNA reduces the size of breast tumours derived from MCF7-Luc in xenotransplants (red dots). ( c) Flow cytometry analyses show that CD47 cell surface protein levels are reduced after knocking down NFKB1 (red histogram) in MCF7 cells. ( b) Knocking down NFKB1 and PPARĪ± by shRNAs reduces CD47 gene expression more than knocking down other candidate transcription factors in the breast cancer cell line MCF7. The binding to E5 of each transcription factor obtained from the MCF7 nuclear extract was compared to the binding to E5 of each transcription factor obtained from HepG2 nuclear extract. Thus, binding of transcription factors to the E5 DNA fragment and not to the consensus sites probes is represented by an increase in RLU while binding to the consensus sites probes and not to E5 is represented by a no change or decrease in RLU. Final RLU (binding of each transcription factor to CD47 E5) was calculated as follows: the average relative luminescence units (RLU) produced by the binding of each transcription factor to the consensus probes when outcompeted with the E5 DNA fragment (binding competition) was subtracted from the average RLU produced by the binding of each transcription factor to the consensus sites probes only (control). A competition assay was performed by incubating the nuclear extract with the E5 DNA fragment and the consensus sites oligos simultaneously. Nuclear extract from MCF7 and HepG2 were incubated with a plate array containing oligos encoding consensus sites for well-characterized transcription factors. ( a) A protein-DNA binding profiling assay reveals that transcription factors NFAT, NFKB, PPAR, SMAD, STAT3, STAT5 and STAT6 bind significantly to E5 in MCF7 (blue bars) and not in HepG2 cells (red bars). 7 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.6 Department of Medical Oncology &Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010, USA.5 Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 USA.4 Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.3 Department of Surgery, Division of Vascular Surgery, Stanford University School of Medicine, Stanford, California 94305, USA.2 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.1 Institute for Stem Cell Biology and Regenerative Medicine, and Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.