ROS Promote Hypoxia-Induced Keratinocyte Epithelial-Mesenchymal Transition by Inducing SOX2 Expression and Subsequent Activation of Wnt/β-Catenin

Figure 4

Validation of the Wnt/β-catenin pathway as a target of SOX2 in HaCaT cells under hypoxia. (a) qRT-PCR was employed to detect the mRNA levels of PORCN, WNT11, FZD5, BAMBI, FOSL1, CTNNB1 (β-catenin gene), LEF1, and LGR5 in HaCaT cells subjected to hypoxia for 12 h. (b, c) Western blotting (b) and quantitative analysis (c) were performed to detect β-catenin, LEF1, and LGR5 levels in cells after 12 h of treatment with hypoxia or normoxia. β-Actin was used as the loading control. (d) Luciferase assays were used to analyse activation of the β-catenin promoter upon SOX2 overexpression in HaCaT cells. (e) ChIP-PCR was employed to uncover the binding of SOX2 to the β-catenin promoter in HaCaT cells. (f) Levels of endogenous ROS were measured by a microplate reader. (g, h) Western blotting (g) and quantitative analysis (h) of β-catenin, Slug, and SOX2 were carried out in HaCaT cells with or without hypoxia and XAV939 treatment for 12 h. (i, j) Scratch assays (i) and quantitative analysis (j) were performed using XAV939-treated and untreated HaCaT cells with or without hypoxia treatment for 12 h. Bar, 200 μm. . . , , , and .

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