Fig 1: ISX expression induces TWIST1 and Snail1 expression, and promotes EMT AThe expression levels of ISX, TWIST1, Snail1, fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (P < 0.001 compared with time point 0 h; Student's t-test) of three independent experiments, each performed in triplicate.BWestern blotting analysis of the protein levels of ISX, HIF1a, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), fibronectin, N-cadherin (N-Cad.), vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8 h after DOX induction.C, DISX transcriptionally activates luciferase activity driven by TWIST1 (C) and Snail1 (D) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.EChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each group was pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.FISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.GChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each group was pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.HEffect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi (described in Materials and Methods). Data are presented as mean ± SD in graph (**P < 0.01, ***P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate. Scale bar, 100 µm.IEffect of forced expression and knockdown of ISX on cell invasion (Transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate. Scale bar, 100 µm. Source data are available online for this figure.