(B) Relative mRNA expression of HIF1-, FOXO3a, N-cadherin, and vimentin under normoxic or hypoxic conditions in PANC-1 and SW1990 tested by RT-PCR. were observed. Hypoxia upregulated the messenger RNA (mRNA) and protein IkappaB-alpha (phospho-Tyr305) antibody expressions of HIF-1, FOXO3a, and the key epithelial-mesenchymal transition (EMT)-related (EMT) molecules N-cadherin and vimentin, as well as the phosphorylation of FOXO3a. Interestingly, hypoxia promoted the extranuclear localization of FOXO3a. Overexpression of FOXO3a not only significantly decreased the invasion, migration, and EMT of PC cell lines, but also reversed hypoxia-induced extranuclear localization. Finally, FOXO3a might act as a tumor suppressor in PC by inhibiting the ERK signaling pathway by inducing DUSP6 expression, and the ERK activator fisetin could effectively attenuate the inhibitory role of FOXO3a on ERK. Conclusions Taken together, our results identified that hypoxia-induced extranuclear localization of FOXO3a promoted cell migration and invasion Bz 423 of human PC by modulating the DUSP6/ERK pathway. migration and invasion assays To determine the cell migration, the transwell assay was performed using a chamber (8 M pore; Corning Life Sciences, Tewksbury, MA, USA) according to the manufacturers instruction. After cells were suspend in serum-free DMEM, they were seeded into the upper Transwell chambers. The lower compartment was placed into 24-well plates which was filled with DMEM with 10% FBS as a chemoattractant. After 24 h incubation, iced methanol was used to fix the cells and then cells were stained with 0.5% crystal violet solution for 30 min at room temperature. After removing the cells remaining in the upper surface of chamber, the number of cells migrated to the lower side were counted by a microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) at 200 magnification. For invasion assays, Transwells were pre-coated with Matrigel (BD Biosciences, USA) before the cells were added. The following steps were the same as those in Bz 423 the migration assay. Immunofluorescence microscopy for FOXO3a detection The PANC-1 cells were seeded in 6-well plates and incubated under normoxic or hypoxic conditions for 24 h. The immunofluorescence staining experiment was performed according to the manufacturers instructions. Briefly, 4% formaldehyde was used to fix PANC-1 cells at room temperature for 30 min. We then used ice-cold Bz 423 absolute methanol to permeabilize the cells at room temperature for 10 min. After blocking by incubation with PBS containing 3% bovine serum albumin (BSA) at room temperature for 30 min, cells were incubated with anti-FOXO3a antibody in blocking buffer for 2 h at room temperature followed by incubation with goat anti rabbit antibody conjugated with Alexa Fluor 594 (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, and nuclei were Bz 423 stained with 1 g/mL DAPI in PBS. Images were taken with a fluorescent microscope (Olympus Corporation, Japan). Plasmid construction and transfection The coding sequence (CDS) of human FOXO3a mRNA were synthesized and digested using restriction enzymes HindIII and EcoRI. After that, the DNA fragment was subcloned into the empty pcDNA3.1 vector to construct the FOXO3a overexpression plasmid. DNA sequencing was used to confirm the integrity of the plasmid constructed. The transfection complex was prepared with Lipofectamine? 3000 (Thermo Fisher Scientific, Inc., USA) for 20 min at room temperature according to the manufacturers instructions, and transfection was carried out at 37 C for 24 h. Western blot assay was performed to determine the expression efficiency of pcDNA3.1-FOXO3a plasmid transfection. Statistical analysis All numerical Bz 423 data were presented as the means standard deviation (SD). GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) software packages were used for all statistical analyses. 2-sided Students although hypoxia exerted a weak inhibitory effect on cell proliferation, both.