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. 2010 Nov 12;5(11):e13971. doi: 10.1371/journal.pone.0013971

Figure 7. The PIAS1-SnoN sumoylation pathway antagonizes TGFβ-suppression of E-cadherin promoter activity.

Figure 7

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 (α-FLAG IP) followed by sequential PIAS1 (α-MYC) and SnoN (α-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 TGFβ-reduction of E-cadherin transcription. NMuMG cells stably expressing SnoN (WT) or SnoN (EdA) or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the PIAS1 protein (+) were subjected to luciferase and β-galactosidase assays (see MATERIALS and METHODS). Each column is the mean (± SEM, n =  6) of percent reduction of E-cadherin-p-luciferase activity by TGFβ. 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 (α-MYC IP) followed by SnoN (α-SnoN) and PIAS1 (α-MYC) immunoblotting. Total lysates were immunoblotted as described in 7A. D) SUMO E3 ligase mutant PIAS1 reverses the effect of SnoN to inhibit TGFβ-repression of E-cadherin promoter activity. NMuMG cells stably expressing PIAS1 (WT) or PIAS1 (CS), or stably transfected with the vector control plasmid (−) were transiently transfected with the E-cadherin-p-luciferase reporter and the β-gal plasmid together with an empty expression vector (−) or one encoding the SnoN protein (+) were subjected to luciferase and β-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).