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. 2016 Mar 14;7(17):23552–23568. doi: 10.18632/oncotarget.8083

Figure 2. KLF8 upregulates CXCR4 at the level of transcription.

Figure 2

(A) Schematic diagram of CXCR4p. Potential KLF8-binding sites (GT boxes) are shown. The CXCR4p was isolated from MDA-MB-231 genomic DNA and inserted in the pGL3basic luciferase reporter vector. (B) KLF8 activates the CXCR4p. The CXCR4p or control vector was co-transfected with KLF8 or activation domain-defective mutant (mKLF8) into NIH3T3 cells. Reporter activity was performed as described in Materials and Methods. (C) KLF8 responsive site is located between −150 and −217 bp of the CXCR4p. Serial CXCR4 truncation mutants were constructed and tested for changes in the promoter activation by KLF8. (D) The GT-box 1 is required for KLF8 to activate the CXCR4p. Indicated GT boxes were mutated (mGT) and tested for changes in the promoter activation by KLF8. (E, F) KLF8 directly binds CXCR4p at the GT-box 1 site. HA-KLF8 was overexpressed in HEK293 cells. The cells were processed for either ChIP assay using primers spanning the GT-box 1 (E) or BOP assay using oligos spanning the wild type GT1 (WT) or its mutant (mGT1) (F) as described in Materials and Methods.