Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment (original) (raw)
Figure 3
Hypoxia and TGF-β increase VEGF and CXCR4 transcription through proximal promoter response elements.
(A) HepG2 cells were transfected with pGL3 plasmids containing full-length for 5′-deleted fragments of the human VEGF or CXCR4 promoter and the phRL-CMV plasmid. Cells were treated ± TGF-β (5 ng/mL) ± 1% O2 during 24 h before measuring dual-luciferase activity. Results are expressed as the mean ± SEM (n = 3) of the relative luciferase activity, analyzed using an unpaired Student's t test. (B) Schematic representation of wild-type (WT), HRE-mutant (mH1) and SBE-mutant (mS1 or mS2) VEGF and CXCR4 promoters. One to three nucleotides (underlined, bold letters) within HRE and SBEs were substituted as indicated. (C) HepG2 cells were transfected with pGL3 plasmids containing a wild-type VEGF or CXCR4 promoter or promoters with mutations to the HRE or SBE and the phRL-CMV plasmid. Cells were treated ± TGF-β (5 ng/mL) ± 1% O2 during 24 h before measuring dual-luciferase activity. Results are expressed as the mean ± SEM (n = 3) of the relative luciferase activity, analyzed using an unpaired Student's t test. * P<0.05, ** P<0.01, *** P<0.005, **** P<0.001.