Figure 3: NFKB1 candidate transcription factor regulates CD47 expression in breast cancer cells.

(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). Nuclear extract from MCF7 and HepG2 were incubated with a plate array containing oligos encoding consensus sites for well-characterized transcription factors. A competition assay was performed by incubating the nuclear extract with the E5 DNA fragment and the consensus sites oligos simultaneously. 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). 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. 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. N=4 samples. Values represent mean±s.d. Student’s unpaired t-test for independent samples was performed. **P<0.01, *P<0.05. (b) Knocking down NFKB1 and PPARα by shRNAs reduces CD47 gene expression more than knocking down other candidate transcription factors in the breast cancer cell line MCF7. N=5 samples. Values represent mean+s.d. Student’s unpaired t-test for independent samples was performed. ***P<0.005, *P<0.05. (c) Flow cytometry analyses show that CD47 cell surface protein levels are reduced after knocking down NFKB1 (red histogram) in MCF7 cells. Grey histogram is the FMO control.