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. 2022 Feb 25;12:801230. doi: 10.3389/fonc.2022.801230

Figure 3.

Figure 3

GDF15 upregulates VEGFA transcription by promoting the binding of SP1 to its promoter. (A) 293T cells were transfected with a luciferase construct containing different VEGFA promotor regions or control vectors and treated with recombinant human GDF15 (rhGDF15). Data are presented as the mean ± standard deviation of three experiments (*p < 0.05). (B) For immunoblotting analysis of MAPK1, p-MAPK1, and SP1 in U373 cells, the cells were incubated for 1 h in the presence or absence of a MAPK1 inhibitor (U0126) and treated with rhGDF15 for 6 h. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of VEGFA mRNA expression in U373 cells treated with rhGDF15 and/or U0126. qRT-PCR analysis was performed using samples extracted 12 h post-rhGDF15 treatment. For inhibition studies, U373 cells were incubated with 10 µM U0126 for 1 h before treatment with 100 ng/mL rhGDF15. (D) Cells were incubated with or without rhGDF15 and U0126 and subjected to chromatin immunoprecipitation (ChIP) assay using anti-SP1 or IgG isotype control antibodies. Bar graphs represent the qRT-PCR results for immunoprecipitated VEGFA promoter. Data are presented as the mean ± standard error (**p < 0.01 and ***p < 0.001 compared with control group; # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 compared with rhGDF15-treated group.