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. 2010 Oct 12;5(10):e13390. doi: 10.1371/journal.pone.0013390

Figure 4. TG2 is a downstream mediator of TGF-β-induced EMT.

Figure 4

(A) Supershift assay for NF-κB activity using EMSA with the nuclear extracts prepared from indicated cells. Nuclear extracts were incubated with an anti-p65 antibody and anti-p50 antibody, or nonradioactive (cold) or mutant NF-κB oligonucleotides and examined for DNA binding. Nuclear extract of KMB cells treated with TNF-α (+TNF) were used in parallel as positive control. (B) Immunoblot analysis showing the level for pAKT(S473) and pFAK(Y397) in 10A-Vec and 10A-TG2 cells. (C) Phase-contrast images of MCF10A cells transfected with control-shRNA or TG2-shRNA and incubated with 2.5 ng/ml recombinant TGF-β for indicated time periods. After 8 and 12 days of TGF-β-treatment MCF10A-control-shRNA cells showed mesenchymal morphology but not the TG2-shRNA transfected cells. (D) Immunoblot analysis for expression of TG2, E-cadherin and fibronectin in MCF10A-control-shRNA and MCF10A-TG2-shRNA cells in response to TGF-β treatment at indicated time points. (E) Downregulation of endogenous (MCF-7/RT) or induced (MCF10A-TG2) TG2 by siRNA resulted in loss of fibronectin (mesenchemyal marker) and upregulation of E-cadherin (epithelial marker) expression. Results shown are from a representative experiment repeated twice with similar results.