Figure 3.

SNAI1 and ZEB-1 directly regulate CD47 in EMT-activated mesenchymal MDA-MB-231 cells and CD47 in human breast cancers is correlated with EMT marker expression.(A-C) MDA-MB-231 cells were transfected with different siRNAs targeting SNAI1, ZEB-1 or scrambled control. (A) SYBR-GREEN RT-qPCR was used to monitor CD47, SNAI1 and ZEB1 mRNA expressions levels. The experiment was performed in triplicate and repeated 3 times. (B) Western blot was performed to show CD47, ZEB1, SNAI1, E-CADHERIN and β-ACTIN protein levels. The experiment was repeated 3 times. (C) Densitometry was performed to compare CD47 protein levels. The experiment was repeated 3 times. (D and E) Surface expression of CD47 on live cells was evaluated by flow cytometry as compared with isotype control (gray-shaded histogram). The experiment was repeated 3 times. (F) Different E-boxes in the Human CD47 promoter (CD47 mRNA, NCBI Reference Sequence: NM_198793.2) are shown. (G and H) ChIP was performed on MDA-MB-231 and MCF7 SNAI1 cells by using either anti-SNAI1 antibody (G) or anti-ZEB1 antibody (H) followed by SYBR-GREEN RT-qPCR using RPL30, E-CAD (E-CADHERIN), E-box sites (E-box-1, E-box-2 and E-box-3) and control primers (a region containing no E-box). For each gene, the RT-qPCR signals were normalized to control IgG. SNAI1 ChIP signal or ZEB1 ChIP signal (fold enrichment over IgG control). Two separate experiments (in triplicate) with the same results were performed. (I and J) Correlation analysis between mRNA expression profiles of CD47 and various EMT marker genes in TCGA (I) and METABRIC (J), 2 large breast cancer data sets, was performed using Pearson's correlation test. *P < 0.05 and ***P < 0.001.