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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

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

Fig. 5

HMGA1 regulates the VEGFA promoter with two independent ways: via Sp1 and FOXM1. a Schematic representation of the VEGFA deletion mutant reporter vectors obtained with progressive deletions of the pGL4.10-VEGFprom (− 500–1) and used in the experiments. FOXM1 binding sites are represented with light blue ovals, whereas the AT-enriched sequences bound by HMGA1 are shown as grey boxes. b Luciferase assay on HEK293T cells transiently co-transfected with the luciferase reporter plasmid pGL4.10-VEGFprom (− 500–1), the deletion mutant vectors pGL4.11-VEGFprom (− 338–1), pGL4.11-VEGFprom (− 172–1) or pGL4.11-VEGFprom (− 104–1) with the expression plasmid pEGFP-HMGA1 (grey bar), pEGFP-FOXM1 (blue bar) and pEGFP-HMGA1/pEGFP-FOXM1 (red bar). pRL-CMV Renilla luciferase expression vector was included to normalize for transfection efficiencies. Values are reported as relative luciferase activity compared to cells transfected with the correspondent reporter vectors and the expression plasmid pEGFP. The data are represented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, NS: Not Significant; two-tailed Student’s t-test. An example of western blot validations is reported in Additional file 8: Figure S5d. c Schematic representation of pGL4.10-VEGFprom (− 500–1) and deletion reporter vectors, pGL4.11-VEGFprom (− 388–1) wild-type (WT) and mutated in SP1-binding sites (MUT). FOXM1 binding site is represented with light blue oval, the AT-enriched sequence bound by HMGA1 is figured as grey box, the orange rhombus represents the SP1 binding element and the black cross indicates the mutation of SP1 binding element. d Luciferase assay on HEK293T cells transiently co-transfected with the luciferase reporter plasmids pGL4.10-VEGFprom (− 500–1), pGL4.11-VEGFpromWT (− 388–1) or pGL4.11-VEGFpromMUT(− 388–1) with the expression plasmids pEGFP-HMGA1(grey bar), pEGFP-FOXM1 (blue bar) or pEGFP-HMGA1/ pEGFP-FOXM1 (red bar). pRL-CMV Renilla luciferase expression vector was included to normalize for transfection efficiencies. Values are reported as relative luciferase activity compared to cells transfected with the reporter vectors used and the expression plasmid pEGFP. The data are represented as the mean ± SD (n = 3). **p < 0.01, ***p < 0.001, two-tailed Student’s t-test. An example of western blot validations is reported in Additional file 8: Figure S5e

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