PIAS1 Negatively Regulates EMT Figure 1. TGFb downregulates the SUMO E3 ligase PIAS1 in cells undergoing EMT. A) E-cadherin immunofluorescence images of NMuMG cells, left untreated (2 TGFb), or incubated with TGFb (+ TGFb), the TGFb-receptor type I kinase (TbRI) inhibitor SB431542 (+ KI), alone or together for 48 h. B) Each column is the mean (6 SEM, n = 3 experiments) of percent suppression of cellular E-cadherin by TGFb relative to that of the basal for each of 2KI and +KI (see MATERIALS and METHODS). TGFb induces EMT as indicated by nearly 90% reduction in the level of the epithelial marker E- cadherin in cells. The KI blocks TGFb reduction of E-cadherin and hence induction of EMT. C) TGFb decreases endogenous PIAS1 levels in NMuMG cells undergoing EMT. Lysates of NMuMG cells left untreated or incubated with TGFb (48 h) were subjected to PIAS1 (a-PIAS1) and actin (a-actin) immunoblotting (IB). D) TbRI-signaling is required for downregulation of PIAS1. Lysates of NMuMG cells left untreated or incubated with TGFb (48 h), KI (for 48 h or for the last 24 h), alone or together, were subjected to PIAS1, E-cadherin (a-E-cadherin), Smad2 (a-Smad2) and phospho-Smad2 (a- pSmad2) immunoblotting. TGFb increases phospho-Smad2 while reducing the levels of PIAS1 and E-cadherin. The KI blocks the ability of TGFb to downregulate PIAS1 and E-cadherin. E) TGFb does not reduce the levels of PIAS1 mRNA. RT-PCR of RNA extracts from NMuMG cells incubated without or with TGFb for 48 h to analyze mRNA levels of PIAS1 and the internal control GAPDH (see MATERIALS and METHODS). F) TGFb enhances endogenous PIAS1 ubiquitination in NMuMG cells. Lysates of cells treated with the proteasome inhibitor MG132 (last 24 h of a 48 h culture), TGFb PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13971