FOXO1 silencing enhances the impact of oxidative stress to reduce keratinocyte migration and increase apoptosis. NHEK cells were transfected with control or FOXO1 siRNAs for 48 h and then treated with 150 µM H2O2, 1 mM NAC, 2 ng/ml TGF-β1, or various combinations of these agents. (A) Intracellular ROS were determined using the fluorescent probe CM-H2DCFDA staining. (B) Quantitative analysis of migrated cells after scratch wounds. (C) Transwell migration assay was carried as described in Materials and methods; the cells that have migrated across the transwells were stained with DAPI and photographed and counted. (D) Apoptotic keratinocytes were measured by TUNEL staining. Quantitative analysis of the percentage of TUNEL-positive nuclei is reported as mean ± SD. The mRNA levels of GPX2 (E), cytoglobulin (F), and GADD45α (G) were analyzed by real-time PCR. Data show mean ± SEM of at least three independent experiments. *, Significantly changed compared with scrambled siRNA; +, significantly changed compared with FOXO1 siRNA in the no treatment group; #, significantly changed compared with the H2O2 plus FOXO1 siRNA. Significance set at P < 0.05.
