Figure 4.

CCN6-mediated E-cadherin repression is dependent on Snail and ZEB1. A: E-cadherin promoter activity of wild-type (left) and E-box mutant (right) constructs was determined in HME cells transfected with the empty vector and HME cells with stable CCN6 knockdown. CCN6-mediated reduction in the activity of the E-cadherin promoter was abrogated by the introduction of mutations in the E-boxes. B: Quantitative SYBR green real-time RT-PCR of the E-cadherin transcriptional repressors SNAIL, SLUG, TWIST1, TWIST2, smad-interacting protein-1, and ZEB1 was performed on HME cells transfected with the empty vector and HME cells with stable CCN6 knockdown. Each sample was tested in duplicate, and a ratio was calculated relative to the housekeeping gene GAPDH. C: Western immunoblot showing that CCN6 knockdown results in a marked up-regulation of Snail and ZEB1 proteins. Snail–E-cadherin localization was determined by confocal laser microscopy. CCN6 siRNA inhibition caused marked nuclear accumulation of Snail protein (red) and loss of E-cadherin (green) protein at the cell membranes compared with the HME vector control cells. D: Snail and ZEB1 are required for CCN6-mediated down-regulation of E-cadherin. siRNA-mediated knockdown of Snail expression decreased Snail protein and increased E-cadherin. In control siRNA-treated cells (si-Co), Snail is increased and E-cadherin is reduced. siRNA-mediated knockdown of Snail expression interrupted the effect of CCN6 down-regulation of E-cadherin expression. Similarly, knockdown with ZEB1 siRNA decreased ZEB1protein and increased E-cadherin levels. siRNA-mediated knockdown of ZEB1 expression interrupted the effect of CCN6 down-regulation of E-cadherin expression.