FIG 8.

The SP1 inhibitor WP631 was used to suppress EMT in HEC cells. (A and B) Noncytotoxic concentrations of WP631 were used in HEC-1 (A) and HEC-6 (B) cells in order to examine in vitro cell invasion. The left graphs show cell viability assayed at each concentration of WP631. The right graphs show quantification of the data. The scale bars represent 200 μm. (C) The mRNA levels of TWIST1 in HEC-1 and HEC-6 cells treated with each concentration of WP631 were examined by real-time RT-PCR. (D) Protein levels of EMT markers, including TWIST, in HEC-1 and HEC-6 cells were analyzed by immunoblotting. (E) The wild-type and mutant pTWIST1+116 reporters were used to examine their responses to WP631 in HEC-6 cells. The control activities of the mutant reporter were adjusted to the same values as those of the wild-type reporter and are shown as white bars. (F) Electrophoretic mobility shift assay showing the inhibition of SP1-DNA complexes by WP631 by use of the nuclear extract from 293T cells. An anti-SP1 antibody was used to confirm that the complexes contained SP1 (supershift). n.s., not significant; *, P < 0.05; **, P < 0.01.