Fig. 1.

Hypoxia promotes HK migration through the action of HIF1. HKs were serum-starved and subjected to two cell migration assays (both 15 hours). (A) Colloidal gold migration assay. Representative images of cell migration tracks are shown together with the migration index (MI). An average migration track under each experimental condition is highlighted with a dotted circle for visual purpose only. (B) The in vitro wound healing assay. Cell migrations were photographed and the remaining cell-free space was quantified as the average gap (AG; double-headed arrows) (Li et al., 2004). Values are the means ± s.e.m. of three independent experiments. (C) Lysates of the cells, which were subjected to hypoxia (1% O₂) or normoxia (20% O₂) for the indicated time, were analyzed by western immunoblotting analysis using antibodies specifically against HIF1α (panels a and c). Anti-GAPDH antibody blotting of duplicate membranes was used as a sample loading control (panels b and d). (D,E) HKs were infected with lentivirus-carrying vector (Vec.), wild-type HIF1α (WT), HIF1αCA (CA) and HIF1αDN (DN). 48 hours following infection, the lysates of the cells were immunoblotted with anti-HIF1α antibodies (D, panel a) or anti-HA tag antibody (E, panel a). Anti-GAPDH antibody was used as a sample loading and densitometry scan control (panel b). (F) The HKs carrying vector or HIF1αWT or HIF1αCA or HIF1αDN were subjected to colloidal gold migration assays under either hypoxia (1% O₂) or normoxia (20% O₂). The computer-assistant quantification of the migration is shown as a migration index, as previously described. ^*Statistically significantly different (P<0.01) from normoxia (20% oxygen). The experiment was carried out four times.