Slug and Snail induction in HSC4 cells in response to TGF-β.

<p><b>(A, B, and C)</b> Slug and Snail mRNA and protein levels in HSC4 cells that had either been treated with 1 ng/ml TGF-β for 24 h or left untreated were determined by qRT-PCR (A and B) and immunoblot analyses (C), respectively. Values were normalized to the amount of <i>GAPDH</i> mRNA (A and B) while α-tubulin was used as a loading control for immunoblotting (C). <b>(D)</b> Invasion assays were performed in HSC4 cells treated with or without 1 ng/ml TGF-β, followed by quantification analyses. The value of the control cells is indicated as “1”. <b>(E)</b> HSC4 cells were treated with 1 ng/ml TGF-β for 24 h, the cells were exposed to docetaxel (DTX; 10 μM). The viable cells were trypsinized and counted using a hemocytometer. The value of the control cells is indicated as “1”. <b>(F and G)</b> Following the siRNA-mediated knockdown of either Slug, Snail, or both in HSC4 cells, the cells were treated with 1 ng/ml TGF-β for 24 h. The levels of Slug and Snail mRNA and protein were determined by qRT-PCR (F) and immunoblotting (G), respectively. mRNA levels were normalized to the amount of <i>GAPDH</i> mRNA (F) while α-tubulin was used as a loading control for immunoblotting (G). <b>(H)</b> After the siRNA-mediated knockdown of either Slug, Snail, or both in HSC4 cells treated with 1 ng/ml TGF-β, the cells were subjected to invasion assays, followed by taking photos and quantification. The value of the control cells is indicated as “1”. <b>(I)</b> After siRNA-mediated knockdown of either Slug, Snail, or both in HSC4 cells treated with 1 ng/ml TGF-β for 24 h, the cells were exposed to docetaxel (DTX) for 24 h. The viable cells were trypsinized and counted using a hemocytometer. The value of the control cells is indicated as “1”. Slug siRNA (#1) and Snail siRNA (#1) were used. Each value represents the mean ± s.d. of triplicate determinations from a representative experiment. Similar results were obtained in at least three independent experiments. NC, non-specific negative control siRNA. <i>p</i> values were determined by Student’s <i>t-test</i>. *<i>p</i> < 0.05, **<i>p</i> < 0.01; n.s., not significant.