Figure 4

Invasive U2OS-M cells have higher levels of DEC2 and accumulate HIF-1α more rapidly in response to hypoxia. A. In vitro migration and invasion activity of U2OS and U2OS-M. Migration and invasion activities were measured in vitro with trans-well chambers, as described in Materials and Methods. Photos are representative fields of invasive cells on the membrane. Magnification: 100 ×. B. Quantification of data shown in A. Bar graphs represent the average number of cells on the underside of the membrane (means ± s.e.m.) C. U2OS-M cells express higher level of DEC2 mRNA than the parental U2OS, while the HIF-1α mRNA level was not significantly affected as determined by qRT-PCR (P > 0.05). D. U2OS-M cells express higher level of DEC2 protein than U2OS cells. Cells were cultured under regular culture conditions or hypoxic condition (1 h) prior to harvest. Protein levels were determined by Western blotting. Under regular culture conditions, neither U2OS nor U2OS-M showed detectable HIF-1α. After 1 h hypoxic exposure, U2OS-M cells, which express higher level of DEC2, accumulated more HIF-1α than U2OS. E. U2OS-M Cells were transfected with either control or DEC2 siRNAs for 48 h. Migration and invasion activities were measured in vitro with trans-well chambers, as described in Materials and Methods. Photos are representative fields of invasive cells on the membrane. Magnification: 100 ×. F. Quantification of data shown in E. Bar graph presents the average numbers of cells on the underside of the membrane (means ± s.e.m.). G. DEC2 knockdown in U2OS-M slowed down HIF-1α accumulation. Cells were transfected with indicated siRNAs and cultured for 48 h. Cells were exposed to hypoxia for 1 h prior to protein sample collection. HIF-1α and DEC2 levels were determined by Western blotting, and β-actin was determined for normalization of loading. ***: P < 0.001.