Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.