Fig. 5: KLF5 upregulates AXL expression via binding to the promoter of AXL.

A, B DCC-2036 downregulated the protein level and mRNA level of AXL. After the MDA-MB-231 cells were treated with DCC-2036 at 10 μM for 48 h, the protein and mRNA levels of AXL were measured by Western Blotting and qPCR. C, D Knockdown of KLF5 decreased the protein level and mRNA level of AXL. MDA-MB-231 cells were transfected with siKLF5 or control siRNA (siNC) for 48 h, followed by WB and qPCR. E Schematic representation of AXL promoter. I–V represented the putative site in the AXL promoter predicted by the Jasper database. One to three represented the qPCR products (fragments) by different AXL primers. F The information of the five highest-scoring putative sites (I–V). G Chromatin immunoprecipitation (ChIP) assay indicated that KLF5 is directly bound to AXL promoter mainly at fragment 2 in MDA-MB-231 cells. The results were shown as relative enrichment compared with IgG. H, I overexpression of KLF5 in MDA-MB-231 cells partially but significantly rescued the DCC-2036-induced reduction of KLF5 binding to fragment 2. MDA-MB-231 cells were transfected with KLF5 or vector control plasmid, followed by treating with 10 μM DCC-2036 for 48 h before WB and ChIP qPCR assay. J Regulation of AXL promoter activity by KLF5 in MDA-MB-231 cells. Wild-type (−2000 to +50 bp) or mutant promoter (site V was deleted) was cotransfected with siNC or siKLF5, and luciferase activity was measured with pRL-TK as an internal control. Data represent the mean of three independent experiments ± s.d. K Regulation of AXL promoter activity by DCC-2036 and overexpression of KLF5 in MDA-MB-231 cells. Wild-type or mutant promoter was cotransfected with KLF5 or vector control plasmid, followed by treating with 10 μM DCC-2036 for 48 h, and then luciferase activity was measured.