Fig. 3

Phosphorylation of FoxM1 at Ser25 facilitates cancer cell migration and invasiveness but not cell proliferation. RFP-tagged wild-type (WT) FoxM1 and S25A, S25E, S361A, S361E, S715A, and S715E mutants were expressed in A549 cells. A549 cells were treated with doxycycline to express RFP-tagged FoxM1. a Immunoblotting was performed using specific antibodies for RFP, N-cadherin (N-Cad), E-cadherin (E-Cad), vimentin, and β-actin (left panel). The band intensity values were quantified using LI-COR Odyssey software, normalized, and plotted (right panel). b qRT-PCR was performed for FOXM1, CDH1, CDH2, and VIM in A549 cells expressing wild-type or mutants FoxM1. *p < 0.05; **p < 0.01; ***p < 0.001; (n = 3). Data are presented as mean ± SD. c Cell proliferation assay was performed (n = 3). Data are presented as mean ± SD of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with experimental control; ^#p < 0.05 compared with indicated groups of cells. d Cells expressing wild-type or mutants of FoxM1 were subjected to a Transwell migration assay. As a positive control for migration, cells were treated with TGF-β. Three days after seeding, the cells on the bottom surface were stained with 0.05% crystal violet dye. Images of the Transwell cell migration assay were collected and analyzed with an Odyssey infrared imaging system and plotted. e An invasion assay was performed using A549 cells expressing wild-type or mutants of FoxM1. Seven days after seeding, the cells that invaded the bottom surface were stained with 0.05% crystal violet dye, and the relative absorbance was plotted. Data are presented as mean ± SD of at least three independent experiments (significantly different from the experimental control). *p < 0.05; **p < 0.01; ***p < 0.001 compared with experimental control. ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 compared with S25A FoxM1