PIAS1 Negatively Regulates EMT Figure 5. SnoN sumoylation contributes to the ability of SnoN to antagonize TGFb-suppression of E-cadherin. A) Wild type SnoN and the SUMO loss of function SnoN (SnoN (KdR)) are expressed at equivalent levels in NMuMG stable cells. Lysates of NMuMG cells stably transfected with the control vector expressing the resistance marker alone or together with wild type SnoN (WT) or SUMO loss of function SnoN (KdR) were subjected to FLAG immunoprecipitation followed by SnoN immunoblotting (upper panel), or were immunoblotted for SnoN and actin, the latter serving as a loading control (lower two panels). B and C) Wild type but not the SUMO loss of function SnoN inhibits TGFb-reduction in E-cadherin protein level in cells undergoing EMT. B) Representative scans of E-cadherin and actin immunoblots of lysates of control vector, wild type SnoN (WT) or SUMO loss of function SnoN (KdR) expressing cells that were left untreated or incubated with TGFb. C) The densities of E-cadherin and actin immunoblots as shown in Figure 5B were analyzed (see MATERIALS and METHODS). Each column in the bar graph represents the mean (6 SEM, n = 4) of percent suppression of E-cadherin protein level by TGFb. D) Representative micrographs of E-cadherin-indirect immunofluorescence of vector control (2), wild type (WT) or SUMO loss of function (KdR) SnoN expressing NMuMG cells that were left untreated (I) or incubated with 20 pM (II) or 100 pM (III) TGFb for 48 h. E) Intensity of E-cadherin immunofluorescence per cell was obtained and analyzed (see MATERIALS and METHODS). Each column in the bar graph represents the mean (6 SEM, n = 3) of percent reduction in E-cadherin levels by 20 pM (clear column) or 100 pM (grey column) TGFb. * indicates statistical significant difference (p,0.05) as compared to the vector control. doi:10.1371/journal.pone.0013971.g005 triplicate, and the data presented represent the mean (6 SEM) of five to six independent experiments. Reverse Transcription-PCR NMuMG cells were lysed using TRIzol lysis reagent (Invitro- gen), followed by total RNA extraction and concentration using chloroform and isopropanol/ethanol, respectively. The reverse transcriptase SuperScript II (Invitrogen) and oligo(dT)12–18 (Amersham Biosciences) as the primer were used to generate Poly(A)-cDNA as described before [38,45,57]. Mouse PIAS1 (1080 bp) and GAPDH (300 bp) cDNA fragments were PCR- amplified using the mouse poly(A)-cDNA as a template and gene specific PIAS1 (sense 59-GGTATACGGGAAAAACCGG-39; antisense 59- TCAGAGGTTACGAGCAAAGG-39), and GAPDH (sense 59-CGGAGTCAACGGATTTGGTCGTAT-39; antisense 59-AGCCTTCTC CATGGTGGTGAAGAC-39) oligo- nucleotides as primers. The PCR products were resolved and stained using 1.2% agarose gels and ethidium bromide, respec- tively. The amplified cDNA products were scanned and visualized using the VersaDoc 5000 Imager (Bio-Rad). Immunoprecipitation and Immunoblotting NMuMG or 293T cell extracts prepared in TNTE (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 1 mM EDTA) buffer containing 0.5% Triton X-100 with protease and phosphatase inhibitors were centrifuged at 14,000 x g for 10 min at 4uC as described previously [38,45,57]. 20 mM N-ethylmaleimide (NEM; Sigma), an isopeptidase inhibitor, was included in the lysis buffer where indicated [38]. Supernatants’ protein compositions were resolved by SDS-PAGE followed by western blotting using antibodies to proteins of interest. In experiments investigating PIAS1 ubiquitination or SnoN sumoylation, the majority of the supernatant was also subjected to immunoprecipitation using goat anti-PIAS1 (N-18; Santa Cruz), or rabbit anti-SnoN (H317; Santa Cruz) antibodies, respectively [38]. In studies examining the association of SnoN and PIAS1, a large fraction of the supernatant was subjected to immunoprecipitation using mouse anti-FLAG (M2; Sigma) or anti-MYC (9E10; Covance). Immunoprecipita- tions were performed on equivalent amounts of total protein in each sample. The protein contents of total cell lysate and immunoprecipitation samples were resolved by SDS-PAGE followed by immunoblotting using rabbit anti-SnoN, rabbit-anti ubiquitin (FL-76; Santa Cruz), mouse anti-E-cadherin (BD Transduction Laboratories), rabbit anti-PIAS1 (Epitomics), mouse anti-Smad2/3 (BD, Transduction Laboratories), rabbit anti- phospho Smad2 (Calbiochem), mouse anti-FLAG, mouse anti- MYC, and rabbit anti-actin (Sigma) as primary antibodies and horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Amersham Biosciences) as the secondary antibodies [57]. ECL (Amersham Biosciences)-generated signals were visual- ized and quantified using a VersaDoc 5000 Imager and Quantity One software (Bio-Rad), respectively. In experiments investigating the effect of expression of wild type PIAS1, SUMO E3 ligase mutant PIAS1 (CS), wild type SnoN, or SUMO loss of function SnoN mutants on the ability of TGFb to inhibit E-cadherin protein expression, the densities of E-cadherin and actin immunoblots in the absence and presence of TGFb were quantified from appropriate scans including those shown in Figures 3B, 5B, and 6D, and E-cadherin levels were normalized to the corresponding actin levels. For each transfectant, reduction of actin-normalized E-cadherin protein levels by TGFb was determined and expressed as a percent of actin-normalized E- cadherin levels in the respective basal (TGFb-untreated) lysates. Each column in the bar graph shown in each of Figures 3C, 5C and 6E represents the mean (6 SEM) of percent suppression of E- cadherin protein levels by TGFb of a transfectant from several independent experiments as indicated by the n value in parenthesis in the respective figure legend. Immunocytochemistry and Fluorescence-Cell Based Analyses 16104 NMuMG cells were seeded per well in 96-well plates and incubated without or with TGFb for 48 h to induce EMT. Cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X100, and blocked using 5% BSA and 5% calf serum in phosphate buffered saline (PBS) [45]. Subcellular localization and levels of E-cadherin were determined by incubating cells with a mouse anti-E-cadherin antibody followed by Cy3-conjugated anti- mouse IgG using a well established indirect immunofluorescence protocol [45]. For whole cell imaging, live cells were stained with 5 mM 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes), according to manufactures instructions, followed by fixing as described above. For actin staining, fixed cells were incubated with TRITC-conjugated phalloidin (Sigma). All cells were incubated with the DNA fluorescent dye Hoechst 33342 (Invitrogen) to visualize their nuclei (data not shown). Images were acquired using the Cellomics Kinetic Scan Reader that is equipped with Carl Zeiss Axiom x microscope and a charge- coupled device (CCD) digital camera [45]. Representative E- cadherin immunofluorescence images are shown in Figures 1A, 3D, 5D, and 6F. Each micrograph corresponds to a 350 mm width. The SpotDetector bioapplication was used to quantify E-cadherin fluorescence intensity per cell, with cells identified by nuclear stain, from images including those shown in Figures 1A, 3D, 5D, and 6F. Each condition was carried out at least in triplicates per experiment, i.e. at least three wells of a 96-well plate, and the immunofluorescence data were averaged from a minimum of 2000 cells per well. For each condition, the reduction of E-cadherin immunofluorescence intensity by TGFb was expressed as a percent of the E-cadherin immunofluorescence level in the PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13971