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. 2012 Nov 21;7(11):e50394. doi: 10.1371/journal.pone.0050394

Figure 6. Prx1 stimulates HIF-1α expression and HIF-1 activity through NF-κB.

Figure 6

A. 2H-11 cells were transfected with the VEGF promoter luciferase reporter construct alone (VEGF Pro) or with a vector encoding for IκB-αSR. Cells were stimulated with 20 nM Prx1 24 hours after transfection and VEGF promoter activity was measured 6 hours later. Results are presented as the average relative VEGF promoter activity; error bars represent SEM. B. HIF-1 activity was assessed using the HIF-1 responsive luciferase reporter. 2H-11 endothelial cells were transfected with the HIF-1 reporter alone (Control) or with an expression vector for an IκB-α SR. Cells were stimulated with 20 nM Prx1 24 hours after transfection and VEGF promoter activity was measured 6 hours later. Results are presented as the average relative VEGF promoter activity. Error bars represent SEM; n≥3. C. 2H-11 endothelial cells were stimulated with rPrx1; cells were harvested at 1 hour. ChIP assays were performed by immunoprecipitation with antibodies to NF-κB and qPCR amplification of isolated DNA fragments with HIF-1α promoter specific primers. Results are normalized to qPCR levels of the HIF-1α promoter prior to enrichment through IP. Results are reported as a fold change to untreated cells. Error bars represent SEM; n≥3. D. 2H-11 endothelial cells were transfected with the HIF-1α promoter (HIF-1α Pro) alone or with an expression vectors encoding for shRNA specific for TLR4 (shTLR4), MyD88DN or IκB-αSR. Cells were stimulated with 20 nM Prx1 24 hours after transfection and HIF-1α promoter activity was measured 6 hours later. Results are presented as the average relative HIF-1α promoter activity; error bars represent SEM; n≥3. ND = not done. D. 2H-11 cells were transfected with NF-κB responsive reporter construct alone (Control) or with vectors encoding for IκB-αSR or shHIF-1α. Cells were stimulated with 20 nM Prx1 24 hours after transfection and NF-κB responsive reporter activity was measured 6 hours later. Results are presented as the average relative NF-κB activity; error bars represent SEM. ** Represents P≤0.01 when compared to control untreated levels; # represents P≤0.05 and ## represents P0.01 when compared to Control stimulated values. N≥3.