Figure 4

CBFβ and Runx2 are required for expression of metastatic genes in MDA-MB-231 cells. (A) CBFβ regulates Runx2-dependent metastatic genes in MDA-MB-231 cells. MDA-MB-231 cells transfected with siCBFβ or a non-specific siRNA were subject to real-time RT-PCR. Total RNAs were used to analyze the mRNA levels of known Runx2 target genes as indicated. Acidic ribosomal phosphoprotein P0 (RPLO) mRNA was used as a control for normalization and relative values are shown. Data are presented as mean ± standard deviation (S.D.) (n = 3). Statistical evaluation of significant differences was performed using the Student's t-test. Asterisk (*) indicates P < 0.05 when compared to control (siNS). (B) Stable knockdown cells (shCBFβ) and the non-specific cells (shNS) were subject to real-time RT-PCR. Data were analyzed as in (A) except that * indicates P < 0.05 when compared to control (shNS). (C) Runx2 regulates metastatic genes in MDA-MB-231 cells. MDA-MB-231 cells transfected with siRunx2 and a non-specific siRNA were subject to real-time RT-PCR. Data were analyzed as in (A). (D) CBFβ is recruited to the OPN promoter in MDA-MB-231 cells. ChIP assays using Runx2 and CBFβ antibodies. The relative enrichment of the OPN sequence was determined by comparing the DNA from specific antibody to that from control IgG then normalized with non-specific genomic DNA. Data are presented as mean ± standard deviation (S.D.) (n = 3). * indicates p < 0.05 compared with IgG by analysis of variance. (E) CBFβ is recruited to the Galectin-3 promoter. ChIP assays using Runx2 and CBFβ antibodies. Data were analyzed as in (D).