Fig. 5.

The sustained activation of signaling pathways and up-regulation of SLUG are required for inducing EMT and invasiveness in breast cancer cells. a and b MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of (5Z)-7-oxozeaenol. a The expression of E-cadherin and vimentin was detected by Western blot. b The cells were used for matrigel invasion assay. c and d MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of SIS3, QNZ, SB203580, PD98059 and SP600125 for 6 days. c The expression of E-cadherin and vimentin was detected by Western blot (left). Relative expression of E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin (right). d The cells were used for matrigel invasion assay. e MCF-7 cells were cultured in presence of TGF-β1 and/or TNF-α for 6 days. The expression of SLUG, SNAIL, ZEB1 and TWIST1 was detected by real-time RT-PCR. f–h MCF-7 cells were transducted with control or SLUG shRNA lentivirus, and then selected for stable expression using puromycin. f The expression of SLUG, E-cadherin, vimentin and β-actin was detected by Western blot. g Relative expression of SLUG, E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin. h The cells were used for matrigel invasion assay. Data are representative of three independent experiments, or pooled from three independent experiments. P values, * P < 0.05, **P < 0.01