Figure 4: Downregulation of AP-2ε mRNA expression in SW1353 cells after treatment with siRNA against HIF1α. (a) HIF1α protein expression under normoxic conditions was compared in mMSC and SW1353 chondrosarcoma cells via western blot. SW1353 cells exhibited much higher physiological HIF1α protein levels (the HIF1α specific band is labeled; cf. Methods, Protein Isolation and Western Blot) and thus were preferred for the following HIF1α knockdown experiment. (b) mRNA expression was analyzed in SW1353 cells after transfection with two siRNA species against HIF1α. Compared to cells transfected with unspecific control siRNA, a significant reduction of the mRNA level of AP-2ε (I) and ANGPTL4 (II) could be determined. Expression of COL2A1 (III) and ACAN (IV) also tended to be reduced while expression of MIA/CD-RAP (V) and SOX9 (VI) was not significantly altered in this experiment. (c) Successful depletion of HIF1α after siRNA treatment was confirmed on mRNA (I) and protein level (II). Numbers indicate densitometric measurement of the intensity of the HIF1α specific band (labeled). (d) To confirm modulation of the transcriptional activity of the HIF1 protein complex the 6xHRE reporter plasmid (cf. Figure 3(c)) was transfected into SW1353 cells which were treated as above. 24 h later luciferase activity was measured and a significant downregulation of promoter activity could be detected with siRNA against HIF1α (data are given as means ± SEM; ns, not significant; ; ; ).