Effect of CCN6 inhibition on the phenotype of mammary epithelial cells. (a) Human mammary epithelial (HME) cells were stably transfected with pSilencer2.1-U6 hygro expression vector expressing CCN6 hairpin siRNA. The suppression of CCN6 was confirmed by reverse transcriptase PCR and Western immunoblotting (WB). HME control cells express low levels of CCN6 protein. C1 and C2 (two stable HME CCN6 siRNA clones) show almost complete inhibition of CCN6. (b) Stable inhibition of CCN6 on HME cells results in a change from compact and oval-shaped cells to irregularly shaped cells with many protruding processes. (c) E-cadherin protein and mRNA were decreased in CCN6-deficient cells. (d) By immunofluorescence CCN6-deficient cells show a drastic decrease of membrane-associated E-cadherin protein. (e) Western immunoblots show that CCN6 inhibition results in a decrease in cytokeratin and an increase in vimentin proteins. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.