Figure 4. TGFb inhibits SnoN sumoylation in cells undergoing EMT. A) Endogenous SnoN is sumoylated in NMuMG cells. NMuMG cell extracts prepared in the absence (2) or presence (+) of the isopeptidase inhibitor N-ethylmaleimide (NEM) were subjected to anti-SnoN or control IgG immunoprecipitation followed by SnoN immunoblotting. Several NEM-sensitive slow migrating SnoN-immunoreactive species as compared to unmodified SnoN were identified (SUMO-SnoN). B) TGFb reduces SnoN sumoylation in cells undergoing EMT. Cells were left untreated or incubated with TGFb for 48 h to induce EMT. NEM- containing lysates were analyzed as described in A. C) Inhibition of TbRI- dependent signaling blocks downregulation of SnoN sumoylation by TGFb. NEM-treated lysates of NMuMG cells left untreated, or incubated with TGFb alone or together with the TbRI inhibitor (KI) were subjected to SnoN immunoprecipitation followed by SnoN immunoblotting as described in A. Each of the scans in A, B and C is a representative blot from an experiment that was repeated two to three times. doi:10.1371/journal.pone.0013971.g004 Materials and Methods Ethics Statement Plasmids CMV-based plasmids to express FLAG-tagged wild type SnoN (WT), SUMO loss of function SnoN where Lysines 50 and 383 were converted to arginine residues (KdR), wild type PIAS1 (WT), and SUMO E3 ligase mutant PIAS1 where Cysteine 350 was PIAS1 Negatively Regulates EMT converted to serine (CS) have been described previously [35,38]. FLAG-tagged SUMO loss of function SnoN (EdA) in which Glutamates 52 and 385 were changed to alanines was generated by a PCR-based approach. The pCAGiP/FLAG expression vector containing cDNA encoding PIAS1 (WT or CS), or SnoN (WT, KdR, or EdA) protein was generated by subcloning the respective cDNA into the pCAGIP vector as described elsewhere [45,56]. A pCAGiP vector enables the coexpression of the gene product of interest and the puromycin resistance marker from an internal ribosomal entry site (IRES) containing bicistronic transcript. The PIAS1 RNA interference vector encoding PIAS1 short hairpin RNAs and enhanced green fluorescent protein (GFP) under the control of the U6 and CMV promoters, respectively, was described previously [38,57]. The E-cadherin-p-luciferase reporter, a generous gift from Dr. A. Cano, and the internal control b-galactosidase reporter constructs have been previously described [45,47,57]. The plasmids were confirmed by restriction digests and/or DNA sequence analyses (University of Calgary Core Sequencing Facility). Cell Cultures and Transfections The NAMRU mouse mammary gland epithelial cells (NMuMG) were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) with high glucose and L-glutamine, supple- mented with 10 mg/mL insulin and 10% fetal bovine serum (Invitrogen). The human kidney epithelial (293T) cells [58,59] were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) with high glucose and L-glutamine, supplemented with 10% fetal bovine serum. NMuMG cells at approximately 70% confluency were incubated in regular media in the absence or presence of TGFb and left at 37uC for 48 h to induce EMT. The TGFb receptor type I kinase (TbRI) inhibitor, SB431542 (Sigma), was used at 10 mM, where indicated. 293T cells were transiently transfected using the calcium phosphate method. NMuMG cells were transiently transfected using FuGENE 6 (Roche Applied Science) or TransIT LT1 (Mirus) according to the manufacturer’s instructions. Pools of NMuMG stables expressing a puromycin- resistant marker alone or together with PIAS1 or SnoN were obtained by transfecting cells with pCAGIP-based vectors containing specific cDNA using Lipofectin (Invitrogen) according to the manufacturer’s instructions, followed by selection of resistant cells using 2 mg/ml puromycin (Invitrogen). Reporter Assays NMuMG cells were seeded in 24-well plates at approximately 4.56104 cells/well one day prior to transfections. Cells were co- transfected with the E-cadherin-promoter-driven firefly luciferase reporter and the CMV-b-galactosidase internal control reporter constructs, together with a control expression vector or one encoding wild type PIAS1 (WT), SUMO ligase mutant PIAS1 (CS), or the PIAS1 RNAi plasmid (PIAS1i), as outlined in the figure legends. 18 h post transfection, cells were incubated in fresh media in the absence or presence of 100 pM TGFb, and left for an additional 48 h, to allow for the induction of EMT where applicable. Lysates were prepared and analyzed for luciferase activity using a commercially available firefly luciferase assay kit [38,45,57]. Arbitrary luciferase activity (relative light units) values were normalized to b-galactosidase activity to account for variations in transfection efficiency. For each transfection, percent repression by TGFb of E-cadherin promoter driven luciferase reporter gene expression was also determined and expressed relative to luciferase activity of the respective basal condition lysates (2TGFb). Each experimental condition was carried out in PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e13971