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. 2016 Aug 24;10(9):1473–1484. doi: 10.1016/j.molonc.2016.08.002

Figure 3.

Figure 3

rBGN stimulates HIF‐1α activity and expression through NF‐kB. (A) VEGF levels in HUVEC culture media were determined via ELISA after cells were stimulated with or without PDTC (or rBGN). (B) HIF‐1α promoter activity was measured in HUVECs that were transfected with a HIF‐1α promoter reporter plasmid (pGL3‐HIF‐1α) in the absence or presence of PDTC (or rBGN). (C) A reporter plasmid for HIF‐1α (pGL3‐HIF‐1α) was generated by cloning the HIF‐1α promoter region (wt) or its NF‐kB binding‐site mutants (mut1and mut2) into the pGL3‐basic vector. rBGN significantly increased the luciferase activity of the HIF‐1α promoter region (wt), while activity was significantly reduced when using mut1 to replace the wt sequence. Mut1 may contain a HIF‐1α‐binding site. (D) Western blotting analysis of NF‐kB, phosphorylated NF‐kB and its downstream protein, HIF‐1α, in HUVECs after cells were stimulated with or without PDTC (or rBGN). GAPDH was used as a loading control. Each value is the mean ± SD of three experiments. ** Represents P ≤ 0.01 when compared to untreated control levels; ## represents P ≤ 0.01 when compared to stimulated control values.