TGF-β1 treatment rescues the migration defect of keratinocytes with FOXO1 knockdown. NHEK cells were transfected with control or FOXO1 siRNAs for 48 h and then treated with vehicle or TGF-β1. (A) Transwell migration assay of cells transfected with control or FOXO1 siRNA; the migrated cells were stained with DAPI and photographed and counted. (B) Quantitative analysis of scratch-wound closure at the indicated time points in FOXO1 knockdown cells without or with TGF-β1. The mRNA levels of integrin α3 (C) and integrin β6 (D) were analyzed by real-time PCR. (E) Cell migration was assessed by transwell migration assay and the cells that have migrated across were stained with DAPI and photographed and counted. (F) ChIP assays for binding FOXO1 to the TGF-β1 promoter were performed and ChIP-enriched DNA was quantified by qPCR with the indicated primers, and values are expressed as percentage of input DNA. (G) NHEK cells were cotransfected with a control pcDNA vector or a vector that expresses constitutively active FOXO1 (FOXO1AAA), control siRNA, or siRNA specific to FOXO1, together with TGF-β1 reporter plasmid. Renilla luciferase reporter was used as an internal control. Luciferase activity was measured 36 h after transfection. Data show mean ± SEM of at least three independent experiments. *, P < 0.05 vs. scrambled siRNA; ⁺, P < 0.05 vs. siFOXO1; ^#, P < 0.05 vs. control pcDNA vector.