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. 2014 Jun 9;18(10):1975–1991. doi: 10.1111/jcmm.12331

Figure 7.

Figure 7

Overexpression of GnT-V in lung cancer cells impairs TGF-β/Smad signalling in a catalytic activity–dependent manner. (A) Overexpression of GnT-V in A549 and H1975 cells impairs TGF-β1-induced phosphorylation of Smad2/Smad3 in a catalytic activity–dependent manner. Vehicle A549/H1975 cells, or those stably overexpressing wtGnT-V or △cGnT-V, were treated with TGF-β1 (5 ng/ml) for 1 hr, the phosphorylation and total protein levels of Smad2/Smad3 were measured by western blot by using anti-pSmad2/anti-pSmad3 and anti-Smad2/3 antibody. (B) Overexpression of GnT-V in A549 cells prevents TGF-β1-induced nuclear translocation of pSmad2/pSmad3 in a catalytic activity–dependent manner. Vehicle, wtGnT-V and △cGnT-V A549 cells were treated with TGF-β1 (5 ng/ml) for 1 hr, then immunofluorescently stained with anti-pSmad2 /pSmad3 antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) Overexpression of GnT-V does not alter TGF-β-non-Smad signalling in A549 and H1975 cells. All cells were treated with TGF-β1 (5 ng/ml) for 1 hr, and signalling molecules including pFAK, FAK, pAKT(Ser473), pAKT(Thr308), pERK, ERK, pP38, P38, pJNK and JNK in TGF-β-non-Smad pathway were determined by western blot. GAPDH was used as loading control. (D) Overexpression of GnT-V in A549 and H1975 cells inhibits TGF-β1-induced Smad2/Smad3 transcriptional reporter activity in a catalytic activity–dependent manner, as determined by a dual-luciferase assay. Vehicle A549/H1975 cells, or those stably overexpressing wtGnT-V or △cGnT-V, were transiently transfected by Smad2/4 driven-3TP-luciferase and Smad3/4 driven-(SBE)4-luciferase. After luciferase reporter vectors' transfection, cells were treated with or without TGF-β1 (5 ng/ml) for another 24 hrs, then dual-luciferase assay was performed. (E) Overexpression of GnT-V in A549 and H1975 cells reduces TGF-β1-induced Snail/Slug expression and nuclear translocation levels in a catalytic activity–dependent manner, as determined by western blot (right) and immunofluorescence staining (left) of A549 and H1975 cells exposed to TGF-β1 (5 ng/ml) for 48 hrs.