Fig 1.
KLF4 promotes VEGF induced cell proliferation.
A, B. Cell proliferation was examined at different time points in KLF4 expressing and knocking down HRMECs. Cells were treated with VEGF or vehicle (Veh) following 24h serum-free media culture before measuring cell proliferation using a MTT assay. Significance was observed between KLF4 expressing and control cells with or without VEGF induction. C. Cell apoptosis was examined using ELISA from HRMECs transduced with lentiviral KLF4shRNA1, 2 and Scramble controls. D. One representative Western blot was shown on the active caspase3 expression at indicated time points in KLF4 knockdown and control cells following 12h serum starvation. Significance was compared between KLF4 knockdown and control cells at the indicated time points. Data are presented as mean ±S.E. from 3 independent experiments, (*p<0.05, **p<0.01).
Fig 2.
KLF4 promotes VEGF induced cell migration.
A, B. Transwell migration assays were performed in HRMECs following KLF4 expression or knockdown. Migrated cells were stained with Crystal Violet and then counted. KLF4 expression in HRMECs significantly increased cell migration while knockdown of KLF4 reduced it as compared to control cells with and without VEGF treatment. The data were presented from three independent experiments in triplicate as mean+S.E and normalized by comparing it to vehicle treated control cells (*p < 0.05, **p<0.01).
Fig 3.
KLF4 promotes VEGF-induced tube formation and enhances angiogenesis in vivo.
A.B. Tube formation assays were performed in KLF4 expressing and knockdown HRMECs, respectively. The angiogenic effect of KLF4 on VEGF induced tube formation was determined by counting nodes and sprouts of tube-like structures from at least three different fields of three independent experiments and normalized to vehicle treated control cells. Significance was compared between KLF4 expressing and control cells with or without VEGF treatment (*p<0.05, **p<0.01, ***p<0.001). Images were presented from one representative experiment. C. Sections of plugs were stained using CD31 antibody and microvessels were counted from 4 sections of each plug and averaged from total 3 plugs. Significance of CD31 positive vessels were compared between sections of KLF4 expressing and control plugs (*p<0.05).
Fig 4.
KLF4 enhances VEGF mediated angiogenesis signaling pathway.
A.B. VEGF signaling pathway was examined in KLF4 expressing and knockdown HRMECs following serum starvation and VEGF treatment using Western blot. Data were presented from three separate experiments by measuring band density using the Image J program. Western blot was shown from one representative experiment. Significance was compared between KLF4 expressing and control cells at different time points of VEGF treatment (*p<0.05, **p<0.01, ***p<0.001).
Fig 5.
KLF4 transcriptionally activates VEGF expression.
A. Luciferase reporter assays were performed to assess KLF4 activation of the VEGF promoter. Luciferase activities in KLF4 expressing and control HRMECs were measured at 24h after transfection with 1.5kb and 0.2kbVEGF promoter luciferase constructs in serum-free conditions. Data were presented as the mean ± SE from three independent experiments. Significance of luciferase activity was found between KLF4 expressing and control cells when 1.5Kb VEGF promoter was transfected in both cells (**p<0.01). B. There are three predicted KLF4 binding sites (CACCC) at the VEGFA promoter. The specific binding sites of KLF4 at the VEGFA promoter was detected by ChIP assay and enrichment of KLF4 binding to sites a and b of VEGFA promoter was significant, not c in KLF4 expressing compared to control cells (*p<0.05,**p<0.01) C: VEGF expression in HRMECs was detected by Western Blot at different time points. Significance was compared to 0h from 3, 6 and 12h (*p<0.05, **p<0.01, ***p<0.001). D: VEGF and KLF4 expression was imaged following immunofluorescence staining at 24h following KLF4 induction. E. VEGF released in cell media was detected using VEGF ELISA assay and significance was observed in KLF4 expressing compared to controls at the indicated time points(*p<0.05). F. Cell migration in KLF4 expressing and control HRMECs following VEGF knockdown using VEGF siRNA was examined using transwell migration assay. (*p<0.05, **p<0.01).
Fig 6.
Schematic diagram of KLF4 mediated VEGF signaling pathway.
KLF4 binds to the VEGF promoter and induces VEGF expression, subsequently phosphorylates VEGFR2 and activates downstream ERK1/2 and AKT to promote cell proliferation, migration and angiogenesis.