Additional file 8: of HMGA1 promotes breast cancer angiogenesis supporting the stability, nuclear localization and transcriptional activity of FOXM1

Figure S5. (a) Schematic representation of the bioinformatic analysis of the 1000 bp VEGFA promoter sequence cloned upstream the luciferase sequence in pGL4.10-VEGFprom (− 1000–1) used in reporter experiments. FOXM1 binding sites are represented with light blue ovals, whereas the AT-enriched sequences bound by HMGA1 are figured as grey boxes. In detailed, we found several AT-rich sequences in the region from – 979 to 907 bp, from − 641 to − 521 bp, from − 355 to − 322 bp, from − 169 to − 75 bp, where the AT stretches are particularly long, and from − 17 to − 13 bp from the TSS of the VEGFA promoter. In addition, we found 9 putative FOXM1 binding sites from − 993 to − 922 bp, from − 643 to – 638 bp, from − 326 to − 322 bp, from − 124 to − 104 bp, where it is located the FOXM1 preferential binding sequence TAAACA, and from − 17 to − 13 bp from the TSS of the VEGFA promoter. (b) Schematic representation of deletion reporter vectors pGL4.10-VEGF (− 1000–500) and pGL4.10-VEGF (− 500–1) obtained from pGL4.10-VEGFprom (− 1000–1). (c) Luciferase assay on HEK293T cells transiently co-transfected with the luciferase reporter plasmid pVEGFprom (− 1000–1), the deletion mutants pVEGFprom (− 1000–500) or pVEGFprom (− 500–1) with the expression plasmids pEGFP-HMGA1 and pEGFP-FOXM1. pRL-CMV Renilla luciferase expression vector was included to normalize for transfection efficiencies. Values are reported as relative luciferase activity comparing to cells transfected with the expression plasmid pEGFP. The data are represented as the mean ± SD (n = 3). NS: not significant; two-tailed Student’s t-test. An example of western blot validations is reported. (d) and (e) Representative images of western blot validations of experiments presented in Fig. 5b and d respectively. (PDF 2000 kb)