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

FIG. 6.

FIG. 6.

β3-endonexin regulates hypoxic HIF-1α transcription via NFκB. (A) HMEC-1 cells were transfected with HIF-1α promoter luciferase constructs containing either the wild-type NFκB-binding site (HIF1α-538) or a mutated NFκB-binding site (HIF1α-538m) and with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) or with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr). Cells were exposed to hypoxia (1% oxygen, Hx) for 8 h or remained under normoxia (Ctr), and luciferase assays were performed. Normoxic controls were set equal to 100% (n=3, *p<0.05 vs. normoxic Ctr/siCtr; #p<0.05 vs. hypoxic Ctr/siCtr). (B) HMEC-1 cells transfected with the HIF-1α promoter luciferase construct (HIF1α-538) were pretreated with the IκB kinase inhibitor BMS-345541 (BMS, 100 μM) and exposed to hypoxia (Hx) for 8 h or remained under normoxia (Ctr), and luciferase assays were performed. Normoxic untreated controls were set equal to 100% (n=3, *p<0.05 vs. normoxic Ctr; #p<0.05 vs. hypoxic Ctr). (C) HMEC-1 cells were transfected with expression vectors encoding β3-endonexin long form (EN-L) or control vector (Ctr). Cells were exposed to hypoxia (Hx) for 8 h or remained under normoxia (Ctr). Chromatin immunoprecipitation assay was performed using an antibody against p65 followed by RT-qPCR with primers amplifying the region of the human HIF-1α promoter containing the NFκB-binding site at −197/188 bp (n=3, *p<0.05 vs. normoxic Ctr; #p<0.05 vs. hypoxic Ctr). (D) HMEC-1 cells were co-transfected with a luciferase construct driven bý the wild-type tissue factor (TF) promoter (pTF636) or the TF promoter mutated at the NFκB site (pTF636NFm) 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).