Figure 2.

STAT3 directly binds to the AXL enhancer and drives its transcriptional activation

(A) Venn diagram illustrating the overlap of predicted AXL regulatory elements identified by four transcription factor-binding site prediction tools: hTFtarget, CHEA, FIMO_JASPAR, and ENCODE. The numbers in each region represent the count of overlapping regulatory sites predicted by one or more tools. Notably, within the central overlap shared by all four databases, two key genes-EGR1 and STAT3 are identified, suggesting their potential roles in the regulation of AXL.

(B) Chromatin immunoprecipitation sequencing (ChIP-seq) signal profiles of STAT3-binding (GSE79707), H3K27ac (histone H3 lysine 27 acetylation), and H3K4me1 (histone H3 lysine 4 monomethylation) across the genomic locus encompassing the AXL gene (GSE29118). Blue peaks indicate regions enriched for STAT3, red peaks represent H3K27ac enrichment, and pink peaks denote H3K4me1 distribution. Black arrows indicate the direction of gene transcription, and blue vertical bars below mark exon positions.

(C) ChIP-qPCR analysis showing the enrichment of STAT3 at the AXL gene enhancer region.

(D) Dual-luciferase reporter assays were performed to assess the transcriptional activity of the AXL enhancer region. Cells were co-transfected with luciferase reporter plasmids containing either the wild-type (WT) or mutant (mut) AXL enhancer sequence, along with either an empty vector (EV) or a STAT3 expression vector.

(E and F) Concurrent RT-qPCR and western blot analyses show that silencing of STAT3 leads to a significant decrease in both AXL mRNA and protein expression compared to the negative control (NC) in HGC-27 cells.

Data are representative of three independent experiments (C–F). Mean ± SD; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 indicate significant difference was assessed using two-tailed Student’s t test.