Fig 2: Knockdown of KLF2 prevents LS-induced reduction of MIF. HUVECs were infected with adenovirus-shKLF2 or -shControl for 48 hours before LS stimulation for 24 hours. The RNA expression of KLF2 (A) and MIF (B) were detected by qPCR. (C) Representative Western blot of intracellular MIF and ß-actin in ECs infected with adenovirus-shKLF2 or -shControl under static culture (ST) or LS conditions. Data shown are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001.

Fig 3: MIF expression is transcriptionally regulated by KLF2. (A) MIF mRNA level in HUVECs determined by qPCR after 48-hour adenovirus-mediated overexpression of KLF2, KLF4 and KLF11 (n = 3). mRNA expression was normalized to HPRT1 mRNA. (B) A total of 2 × 105 cells were counted and seeded into each well for the following MIF ELISA assay. HUVECs were infected with Ad-GFP, Ad-KLF2 for 2 h followed by incubation in fresh medium for 48 h. MIF secreted by HUVECs was measured by ELISA after another 48-hour incubation in serum-free medium (n = 6). (C) Representative Western blotting (top) and the corresponding quantitative analysis of band intensity (bottom) show the expression of intracellular MIF protein relative to the loading control ß-actin in ECs overexpressing KLF2, KLF4, KLF11 or GFP (n = 3). (D) Dual luciferase activity was assessed in Ad293 using the 1-kB MIF promoter driven Luciferase construct and the mutated one in combination with expression plasmids for KLF2. Mutation of the indicated binding sites was performed using the Site-Directed Mutagenesis kit. Data represent relative luciferase activity (normalized to Renilla luciferase), n = 6 per group. (E) HUVECs were infected with adenovirus-KLF2 or -GFP for 72 hours. ChIP assay was performed using a rabbit antibody against KLF2 and equivalent amount of rabbit IgG as control. The binding of KLF2 to the MIF promoter was determined by qPCR. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001.