Figure 2.

PROX1 binds to the MMP14 promoter and regulates its expression. (a) Upper panel: schematic diagram of the MMP14 promoter fragments, numbers indicate the bp upstream (−) or downstream (+) of the MMP14 transcription start site (TSS, where bp = 0 is MMP14 TSS), indicated by the black arrow. Bottom panel: luciferase reporter assay using either the pGL3 backbone or its indicated derivatives depicted above. HeLa cells were co- transfected with the indicated reporter plasmids (0.1 µg) and expression vectors for PROX1 WT or MUT (1 µg) in duplicates. An empty pAMC vector was used as a control (mock). 32 h after transfection, cell extracts were collected and luciferase activities were measured. The graph includes the data from three independent experiments. Error bars represent SD. (b) Upper panel: schematic representation of the MMP14 promoter region spanning from bp −1340 to −36. Letters denote the fragments amplified by different sets of primers (a–h) used in the ChIP-qPCR below. Bottom panel: Chromatin immunoprecipitation using either PROX1 or control IgG antibodies followed by qPCR with primers amplifying the indicated regions of the 1.2 kb MMP14 promoter region. Average fold enrichment over the IgG is shown for three independent experiments; error bars represent SD. (c) Left: schematic representation of the 1.2 kb MMP14 promoter reporter plasmid, the b and c regions immunoprecipitated in (b) are marked as red boxes. PROX1 binding site containing regions (BS1 and BS2) are illustrated below as red dotted lines, numbers indicate their position upstream of the MMP14 TSS. These sequences were deleted in the 1.2 kb MMP14 promoter reporter plasmid (white boxes), generating the 1.2 kb MMP14 promoter mutants ΔBS1, ΔBS2, ΔBS1-2. Right: Luciferase assay performed as in (a) using 1.2 kb MMP14 promoter either full length (FL) or the indicated mutants co-transfected with either a pAMC control vector (mock) or a PROX1 WT expression vector. *p < 0.05; **p > 0.01; ***p > 0.001.