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. 2009 Dec;11(12):1318–1328. doi: 10.1593/neo.91084

Figure 6.

Figure 6

Twist transcriptional regulation of CD24 expression. (A) Schematic representation of the CD24 promoter sequence showing the location of putative TWIST binding sites relative to the transcription start site (+1). Tick marks denote the canonical E-box sequences (CANNTG) to which Twist can potentially bind. The numbers above the tick marks indicate the relative position of E-box sequences within the promoter region. (B) Reporter assays were performed using CD24 promoter-reporter constructs in MCF-7 with exogenous Twist expression plasmid added and estimated for 2 days. The results of the promoter assays are represented as a histogram showing normalized luciferase readings in CD24 promoter activity. (C) Immunoblot analysis of cell extracts from MCF-7 and MCF-7/Twist cells scored for CD24 (Santa Cruz Biotechnology) and Twist protein expression. Actin was used as a loading control. (D and E) In vivo binding of Twist protein to the CD24 promoter sequence. ChIP was carried out using MCF-7/Twist cells and analyzed using CD24 promoter-specific primers by PCR. Identical volumes from the final precipitate were used for the PCRs except for the input, which was diluted 10-fold.