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. 2014 May 1;20(13):1964–1976. doi: 10.1089/ars.2013.5286

FIG. 4.

FIG. 4.

β3-endonexin decreases mRNA levels of HIF-1α and its target genes under hypoxia. (A–C) HMEC-1 cells were transfected with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr) and exposed to hypoxia for 1, 4, and 8 h or cultivated under normoxia (0 h). RT-qPCR analyses were performed with primers amplifying human HIF-1α (A), VEGF (B), or GAPDH (C) as well as 18S rRNA for normalization. Control levels at normoxia (siCtr, 0 h) were set equal to 1 (n=3, *p<0.05 vs. siCtr 0 h; #p<0.05 vs. siCtr 1, 4, or 8 h). (D) HMEC-1 cells were co-transfected with a luciferase construct driven bý the wild-type PAI-1 promoter (PAI-796) or the PAI-1 promoter mutated at the HRE (PAI-796m) and expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr) or with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr). Cells were exposed to hypoxia (Hx) for 8 h or remained under normoxia (Ctr), and luciferase assays were performed. Normoxic controls (Ctr) were set equal to 100% (n=3, *p<0.05 vs. normoxic Ctr/siCtr; #p<0.05 vs. hypoxic Ctr/siCtr). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAI-1, plasminogen activator inhibitor-1; siRNA, short interfering RNA; VEGF, vascular endothelial growth factor.