Downregulation of ZO-1 or overexpresion of ZONAB induces expression of EMT markers.
RT-PCR amplification of mesenchymal markers (A) on RNA isolated from laser captured RPE cells (B) from eyes collected 10 days after subretinal injection with either LNT.shGFP, LNT.ZONAB or LNT.shZO-1. Specific primers for GFAP, vimentin, N-cadherin, cyclin D1, Snail1, CtBP1 and α-SMA were used as described in Materials and Methods. EMT markers associated with cell cycle progression such as Snail1 and cyclin D1 were found to be upregulated in either LNT.ZONAB or LNT.shZO-1 treated eyes. EMT morphological markers such as GFAP, vimentin and N-cadherin were also found to be upregulated in eyes showing overexpression of ZONAB or downregulation of ZO-1. (B) Indicative image of selected RPE cells before (upper image) and after (lower image) laser capture microdissection. Approximately 100 cells (three times the area indicated) were collected per eye per treatment group. Size bar, 20 µm. n = 4 per treatment group.