Twist1 small interfering rna sirna

The entire sequence of the complementary DNA (cDNA) of Twist1 was generated using the total cDNA of normal human embryo. The latter was digested with XhoI/EcoRI, and subcloned into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA). The resulting constructs were verified by DNA sequencing. Twist1 small interfering RNA (siRNA), Opti-MEM medium, control siRNA, and siRNA transfection reagent were purchased from Santa Cruz. Crystal violet, 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), and Rh123 were purchased from Sangon Biotech (Shanghai, China). Matrigel and transwell chambers were purchased from Biosciences (San Jose, CA, USA). ActinRed and Annexin V-FITC Apoptosis Detection Kit were purchased from KeyGEN BioTECH (Nanjing, China). Human recombinant TGF-β1 (rhTGFβ1) and anti-TGF-β1 neutralizing antibody was purchased from R&D Systems (Minneapolis, MN). The antibodies to ABCB1, ABCC1, vimentin, E-cadherin, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Affinity Bioreagents (Colorado, USA). Annexin V-fluorescein isothiocyanate (FITC)-goat anti-mouse IgG and tetramethylrhodamine (TRITC)-goat anti-rabbit IgG were purchased from EarthOx (San Francisco, USA).