Twist expression in HCC cell lines. A, B. Cells were grown to confluence in 6-well tissue culture plastic dishes. Total RNA was extracted from various HCC cell lines. mRNA expression of twist and GAPDH were determined by RT-PCR using a twist-specific primer set (A). Shown are representative gel pictures of three independent studies. Twist protein expression was analyzed by SDS-PAGE and immunoblot using anti-twist antibody (B). Immunoblotting for α-actin expression levels was used to verify equal loading of cellular protein. Normal liver samples were used to determine twist expression level in nonmalignant tissue. Shown are representative blots of more than three independent studies. C, D. Cells were plated and quiesced for 24 h in DME with 0.1% dialyzed FBS. Later the cells were cultured with (black bars) or without (clear bars) 10% FBS. Cell proliferation (C) and cell migration activities (D) were assessed as described in Materials and Method. All results are expressed as a ratio to non-treated HLE cells. The data are the mean ± SEM of at least three independent studies each performed in triplicate.