Figure 5: The hypoxia-induced up-regulation of integrin β1 promoted HaCaT cells migration.

(A) The following experiments are shown: HaCaT cells exposed to no treatment (Control), integrin β1 was upregulated following the inhibition of Notch1 signaling using DAPT (DAPT), integrin β1 was upregulated by low oxygen tension (Hypoxia), cells transfected with a negative control (siRNAcon) or a siRNA against integrin β1 (siRNA ITGB1) were scratch-wounded using Culture-Inserts, whose width of cell-free gap is 500 μm. The results were recorded using a phase-contrast microscope connected to a digital camera from time 0 to 12 h (n = 4 independent experiments). Bar = 200 μm. Before 12 h monitoring, each group was treated with indicated treatment for 12 h. As to siRNA groups, 6 h for interference, 12 h cultured under indicated condition. (B,C) Wound closure was demonstrated by determining the area covered by keratinocytes immediately after wounding and 12 h later. Each panel represents the wound closure level of each group. The results were calculated by measuring the reduction in the wound bed surface over time using Image J software. *P < 0.05 versus control group. ^#P < 0.05 versus the group including siRNA con. ^##P < 0.05 versus hypoxia group. (D) Western blot showing the expression of integrin β1 after interference with siRNA ITGB1 in HaCaT cells under normoxia (left panel) and hypoxia (right panel) conditions. (E) HaCaT cell proliferation was analyzed using Cell Counting Kit-8 (n = 4 independent experiments). (F) Schematic model of oxygen tension tightly regulating the expression of integrin β1 and keratinocyte migration through Notch1 signaling. After injury, high oxygen consumption and vascular injury result in partial hypoxia. Low oxygen tension in wound edge induces keratinocytes to express integrin β1 via inhibition of Notch1 signaling. Integrin β1, as a lamellipodia protein, improves keratinocytes migration during the re-epithelization process.