Fig. 3: The CXCL5/CXCR2 induces the expression of VEGF-A dependent on FOXD1.

a Western blot analyses of HIF-1α, C-JUN, and FOXD1 expression in the different groups. b A heat map displays some FOX protein expression level after being treated with or without rhCXCL5 in HUVECs. c Fold changes of the relative mRNA level of VEGF-A-related FOX gene after being treated with or without rhCXCL5 in HUVECs. d Images of HUVEC tube formation assay in each group. Scale bar, 200 μm (magnification ×40). e, f Number of tubes and total length of tubes in different groups, FOXD1 silencing in HUVECs significantly decreases HUVEC tube formation. g, h VEGF-A protein expression is examined by western blot and ELISA in different groups. i ChIP-qPCR assay using Flag antibody or control IgG in HUVECs transfected with a FOXD1 (Flag-tagged) plasmid shows the binding of FOXD1 on the VEGF-A promoter. j A reporter plasmid for VEGF-A (pGL3-VEGF-A) was generated by cloning the VEGF-A promoter region (WT) or its FOXD1 binding site mutants (MT) into the pGL3 basic vector. rhCXCL5 significantly increased the luciferase activity of the VEGF-A promoter region (WT), while the activity was significantly decreased when transfected with MT sequence. Meanwhile, VEGF-A luciferase activity was inhibited when HUVECs were transfected with the shFOXD1 plasmid. k Images of transwell assay in each group. Scale bars, 100 μm (magnification ×100). l Migration cell numbers were reduced by knocking down FOXD1 in HUVECs. m Images of the EdU assay in each group. Scale bars, 200 μm (magnification ×40). n Proportion of cells in the S phase were reduced by knocking down FOXD1 in HUVECs. Data represent the mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001