Fig. 4

Wound-healing assays in A549 cells transfected with miR-200c mimic and siRNA against HIF-1α. a and b – In these assays, we used A549 cells because the cell migration of NCI-H460 cells was very slow under our experimental conditions. A549 cells were transfected with miR-200c mimic and siR783 (20 nM). After a scratch was made, the cells were grown for 43 h in normoxia or hypoxia. Images were taken 0 and 43 h after wound formation (a) and cell migration toward the wound region was measured (b). ***p < 0.001. c – Efficient knockdown of HIF-1α by siR783, as revealed via western blot analysis. In the wound-healing assays, we used siR783 as an siRNA that targets HIF-1α because siR2210 inhibited cell migration even in normoxia, most likely due to off-target effect. (−): no treatment with CoCl₂; (+): treatment with CoCl_2. d and e – Rescue experiments. A549 cells were first transfected with NC or miR-200c and 6 h later, transfected with pcDNA3.1 or pcDNA3.1-HIF-1α. After 43 h in normoxia or hypoxia, cells underwent cell migration evaluation (d) and western blot analysis (e). In (e), the notations are 1, 4: co-transfected with NC and pcDNA3.1; 2, 5: co-transfected with miR-200c and pcDNA3.1; 3, 6: co-transfected with miR-200c and pcDNA3.1-HIF-1α. For western blot analyses, the pcDNA3.1 and pcDNA3.1-HIF-1α plasmids were transfected at 70 ng/2 ml in a 6-well plate. **p < 0.01