Figure 3: Upregulation of AP-2ε mRNA expression in mMSC after treatment with DP and DFX. (a) mMSC were treated with the hypoxia-mimicking iron chelators 2,2′-dipyridyl (DP) and desferrioxamine (DFX) at a concentration of 100 and 250 μM. Respective controls were incubated with solvent only. 24 h later total RNA was isolated and mRNA expression was analyzed via qRT-PCR. Compared to the controls, a significant upregulation in the expression of AP-2ε (I) as well as of the positive control Angptl4 (II) was detected after DP/DFX treatment. Expression of Col2a1 (III) and Acan (IV) also tended to be enhanced while expression of MIA/CD-RAP (V) was completely unaffected by DP/DFX. Further, a significant induction of Sox9 (VI) expression could be determined. (b) HIF1α protein accumulation due to the chemical compounds was confirmed by western blot analysis. Numbers indicate densitometric measurement of the intensity of the HIF1α specific band (labeled). (c) Enhanced HIF1 transcriptional activity was shown by transfection of a reporter plasmid containing six consecutive HRE binding motifs (6xHRE) into mMSC which were treated as above. 24 h later luciferase activity was measured and a significant upregulation of promoter activity could be detected after incubation with DP/DFX. (d) Additionally, the activity of a 604 bp human AP-2ε promoter construct (AP-2prom604) was measured in the cells. DP/DFX treatment for 24 h did not result in an upregulation of its activity (data are given as means ± SEM; ns, not significant; ; ).