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Figure 4


Fig. 4. Depletion of HIF1{alpha} inhibits keratinocyte cell migration. (A) HaCaT keratinocytes transfected with a nonrelevant siRNA (siCtrl) and a siRNA targeting HIF1{alpha} (siHIF1{alpha}) were scratch wounded with a micropipette tip and filmed for 24 hours using time-lapse videomicroscopy. The wound closure is illustrated by showing the wound size (arrow and a) immediately after injury and 12 and 24 hours after wounding. The right panel represents the quantification of the siHIF1{alpha} effect on the wound closure calculated by the measure of the diminution of the wound bed surface upon time by using Image J software. Results are represented as the percentage of the wound closure of monolayers obtained with cells transfected with the siHIF1{alpha} compared with that observed in the cultures of cells transfected with the nonrelevant siRNA (siCtrl) (taken as the 100% value). Each experiment was performed at least three times, and different areas of several scratches were randomly taken. (B) siRNA-transfected HaCaT keratinocytes were seeded in the upper chambers of Transwell inserts. After 24 hours, cells on the lower side of the filter were stained with DAPI and scored in five independent fields. Experiments were carried out three times and each condition was performed in triplicate. (C) Verification of HIF1{alpha} abrogation by the specific siRNA duplex. Transfected cells were scratch wounded, harvested 6 hours later then HIF1{alpha} mRNA and protein were analysed respectively by real time Q-PCR (left panel) and by western blotting (right panel).





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