PIAS1 Negatively Regulates EMT Figure 6. Sumoylation mediates the ability of SnoN to antagonize TGFb-suppression of E-cadherin expression. A) A schematic showing the two SUMO consensus motifs in SnoN and the amino acid residues that are mutated in each of the SnoN (KdR) and SnoN (EdA). B) Glutamates 52 and 385 are critical for SnoN to be sumoylated on Lysines 50 and 383. NEM-treated lystaes of 293T cells expressing HA-tagged SUMO1, alone or together with SnoN (WT), SnoN (EdA) or SnoN (KdR) were subjected to FLAG immunoprecipitation followed by sequential HA (a-HA) and SnoN immunoblotting. Representative scans from an experiment that was done 4 times. C) Equivalent expression of SnoN (WT) and the SnoN (EdA) in NMuMG stable cells. Lysates of cells stably transfected with the control vector, SnoN (WT) or SUMO loss of function SnoN (EdA) were subjected to FLAG immunoprecipitation followed by SnoN immunoblotting, or were immunoblotted for SnoN and actin. D and E) SnoN (WT) but not SnoN (EdA) inhibits TGFb-reduction in E-cadherin protein level in cells undergoing EMT. D) Scans of E-cadherin and actin immunoblots of lysates of control vector, SnoN (WT) or SnoN (EdA) expressing cells that were left untreated or incubated with TGFb as in Figure 3B. E) Each column is the mean (6 SEM, n = 5) of percent suppression of E-cadherin protein by TGFb obtained by analysis of immunoblots including those shown in 6D (see MATERIALS and METHODS). F) Representative images of E-cadherin immunofluorescence of vector control (2), SnoN (WT) or SnoN (EdA) expressing NMuMG cells that were left untreated (I) or incubated with TGFb (II and III) as in Figure 3D. G) Each column is the mean (6 SEM, n = 5) of percent reduction in E-cadherin levels by 20 pM (clear column) or 100 pM (grey column) TGFb from images as shown in 6F (see MATERIALS and METHODS). * indicates statistical significant difference (p,0.05) as compared to the vector control. doi:10.1371/journal.pone.0013971.g006 respective basal (TGFb-untreated) cells. Each column in each bar graph in Figures 1B, 3E, 5E, and 6G represents the mean (6 SEM) of percent suppression of E-cadherin immunofluorescence intensity by TGFb of a transfectant from several independent experiments as indicated by the n value in parenthesis in the respective figure legend. Figure 7. The PIAS1-SnoN sumoylation pathway antagonizes TGFb-suppression of E-cadherin promoter activity. A) SUMO loss of function SnoN associates with PIAS1. Lysates of 293T cells expressing PIAS1, SnoN (WT) or SUMO loss of function SnoN (EdA) mutant, alone or together were subjected to SnoN immunoprecipitation (a-FLAG IP) followed by sequential PIAS1 (a-MYC) and SnoN (a-SnoN) immunoblotting. Expression of PIAS1, SnoN, and actin in lysates were confirmed by MYC, SnoN, and actin immunoblotting. B) The SUMO loss of function SnoN mutant inhibits PIAS1 to block TGFb-reduction of E-cadherin transcription. NMuMG cells stably expressing SnoN (WT) or SnoN (EdA) or stably transfected with the vector control plasmid (2) were transiently transfected with the E-cadherin-p-luciferase reporter and the b-gal plasmid together with an empty expression vector (2) or one encoding the PIAS1 protein (+) were subjected to luciferase and b-galactosidase assays (see MATERIALS and METHODS). Each column is the mean (6 SEM, n = 6) of percent reduction of E-cadherin-p-luciferase activity by TGFb. C) The SUMO ligase mutant PIAS1 interacts with SnoN. Lysates of 293T cells expressing SnoN, PIAS1 (WT) or SUMO E3 ligase mutant PIAS1 (CS), alone or together were subjected to PIAS1 immunoprecipitation (a-MYC IP) followed by SnoN (a-SnoN) and PIAS1 (a-MYC) immunoblotting. Total lysates were immunoblotted as described in 7A. D) SUMO E3 ligase mutant PIAS1 reverses the effect of SnoN to inhibit TGFb-repression of E-cadherin promoter activity. NMuMG cells stably expressing PIAS1 (WT) or PIAS1 (CS), or stably transfected with the vector control plasmid (2) were transiently transfected with the E-cadherin-p- luciferase reporter and the b-gal plasmid together with an empty expression vector (2) or one encoding the SnoN protein (+) were subjected to luciferase and b-galactosidase assays. Data from 6 independent experiments are presented as outlined in Figure 7B. * indicates statistical significant difference (P,0.05) as compared to vector control (column one of each B and D). doi:10.1371/journal.pone.0013971.g007 PLoS ONE | www.plosone.org 12 November 2010 | Volume 5 | Issue 11 | e13971