Fig. 3

AMD1 positively correlates with Sox10 and is a direct transcriptional target of Sox10. (A) Analysis of TCGA and MEBTABRIC datasets for the expression of AMD1 and Sox10. The relative level of AMD1 was plotted against that of Sox10. (B) Box-plots indicated Sox10 mRNA expression in different subtypes of breast cancer from TCGA and MEBTABRIC datasets. (C) Expression of AMD1 and Sox10 was analyzed by quantitative real-time PCR in SUM159 and Hs578T cells infected with empty vector or Sox10-expressing vector. *p < 0.01 by Student’s t-test. (D) Expression of AMD1 and Sox10 was measured by immunofluorescent staining in SUM159 cells infected with empty vector or Sox10-expressing vector. Nuclei were visualized with DAPI (blue). Scale bar = 30 μm (right). (E) Expression of AMD1 and Sox10 was examined by western blotting in SUM159 and Hs578T cells infected with empty vector or Sox10-expressing vector. (F) Schematic diagram showed positions of potential Sox10-binding motifs in AMD1 promoter. AMD1 promoter luciferase construct and mutated derivatives were also displayed. Sox10 consensus sequence: (A/T)(A/T)CAA(A/T)G. (G) AMD1 promoter luciferase construct (FL1) was co-expressed with empty vector or Sox10-expressing vector in SUM159 and Hs578T cells, respectively. After 48 h, luciferase activities were analyzed (mean ± SD in three separate experiments). (H) AMD1 promoter luciferase constructs (FL1, FL2, FL3 and FL4) were co-expressed with empty vector or Sox10-expressing vector in HEK-293T cells. Luciferase activities were analyzed as in (G). (I) AMD1 promoter luciferase construct (FL1) as well as its mutants (mut1 and mut4) were co-expressed with empty vector or Sox10-expressing vector in HEK-293T cells. Luciferase activities were analyzed as in (G). (H) ChIP analysis for binding of Sox10 to the AMD1 promoter in SUM159 and Hs578T cells