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Fig. 6. Manipulation of N-cadherin expression in NMuMG/E9 cells did not interfere with TGF-ß1-induced morphological change. (A) N-Cadherin-knockdown NMuMG/E9 cells (NMuMG/E9Ncad cells) showed similar morphology to parental NMuMG/E9 cells in phase-contrast microscopy (a,b). Photographs were taken of living cells using a 10x phase objective; scale bar, 50 µm. Parental NMuMG/E9 cells and NMuMG/E9Ncad cells were stained with N-cadherin mAb in the absence of TGF-ß1 (c,d). Cell-cell border staining of N-cadherin was significantly reduced. Photographs were taken using a 40x dry objective; scale bar, 10 µm. (B) TNE extracts of NMuMG/E9 cells and NMuMG/E9Ncad cells in the absence and presence of TGF-ß1 (5 ng ml^–1 for 1 day) were immunoblotted for N-cadherin, E-cadherin, GAPDH and fibronectin. N-Cadherin and E-cadherin levels were quantified and normalized to GAPDH as shown on the bar graph. (C) NMuMG/E9Ncad cells in the absence (a,c) or presence of TGF-ß1 (5 ng ml^–1 for 1 day; b,d) were stained for paxillin (a,b) and F-actin (c,d). Photographs were taken using a 40x dry objective; scale bar, 10 µm. (D) Transwell motility assays were done using control knockdown NMuMG/E9 cells (ctr KD) and NMuMG/E9Ncad cells in the absence of TGF-ß1 showed that NMuMG/E9Ncad cells were significantly less motile than control cells (P<0.05). (E) NMuMG/E9 cells expressing 2x-Myc-tagged human N-cadherin (NMuMG/E9 Ncad cells) showed similar morphology to parental NMuMG/E9 cells in phase-contrast microscopy (a,b). Photographs were taken using a 40x dry objective; scale bar, 10 µm. (F) TNE extracts of parental NMuMG/E9 cells and NMuMG/E9 Ncad cells were immunoblotted for N-cadherin (top) and E-cadherin (bottom).

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