Figure 5. SMURF1 overexpression in NIH-3T3 cells leads to loss of contact inhibition. (A) Transfection of SMURF1 cDNA, or a catalytic mutant of SMURF1 (C699A) (SMURF1m), leads to overexpression (by Western blot) compared to empty vector control. SMURF1 overexpres- sion levels, normalized to GAPDH, are indicated. (B) SMURF1 (but not SMURF1m) overexpression in NIH-3T3 cells leads to loss of contact inhibition (i.e. increased foci formation). Assays done in triplicate (mean +/2 1SD shown); *, P,0.05 (Student’s t-test). doi:10.1371/journal.pone.0023924.g005 siRNA transfections On-TARGETplus siRNAs targeting SMURF1, along with a negative control siRNA pool (ON-TARGETplus siCONTROL Non-targeting Pool), were obtained from Dharmacon (Lafayette, CO). Sequences of siRNAs are listed in Table S1. AsPC-1 cells were grown at 37uC in complete media of RPMI-1640 (Invitro- gen, Carlsbad, CA), 10% FBS, 50 U/ml penicillin, and 50 U/ml streptomycin. For transfection, 150,000 cells were seeded per 6- well plate well, and transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were transfected with a final concentration of 50 nM siRNA for 6 hrs. Western blot Cells were lysed in 16 RIPA buffer supplemented as described [6]. Forty mg total protein lysate was electrophoresed on a 4–15% polyacrylamide gel, then transferred to PVDF membrane and blocked in TBST-T with 5% dry milk. Antibodies were used as follows: anti-SMURF1 rabbit polyclonal antibody (H-60; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution; anti- GAPDH rabbit polyclonal antibody (Santa Cruz Biotechnology) at SMURF1 Amplification in Pancreatic Cancer 1:5,000 dilution; HRP-conjugated anti-rabbit IgG (Pierce, Rock- ford, IL) at 1:20,000 dilution. Detection was done using an ECL kit (GE Healthcare, Piscataway, NJ), and intensities quantified by densitometry using Scion Image software (Scion Corporation, Fredrick, MD). Cell growth and invasion assays Cell proliferation/viability was quantified by colorimetry based on the metabolic cleavage of the tetrazolium salt WST-1 in viable cells, according to the manufacturer’s protocol (Roche, Indianap- olis, IN). BrdU incorporation was determined by colorimetric ELISA using the BrdU Cell Proliferation Assay, according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA). Invasion was quantified by Boyden chamber assay (BD Biosciences, San Jose, CA). Briefly, 24 hrs after transfection, 50,000 cells were plated into 24-well Matrigel-coated inserts with a 0.5% to 10% FBS gradient. Seventy-two hrs later, cells were fixed, stained with crystal violet, and cells traversing the membrane counted. All assays were done as triplicate transfections, and all experiments were repeated at least once with similar results. TGFb transcriptional reporter assay Cells were co-transfected with 4 mg p3TP-Lux (Addgene, Cambridge, MA), a TGFb responsive firefly luciferase reporter containing three consecutive TPA response elements (TREs) and a portion of the plasminogen activator inhibitor 1 (PAI-1) promoter region [17], along with 0.4 mg pRL-TK (Promega, Madison, WI) expressing Renilla luciferase as an internal normalization control. Luciferase activity was assayed 48 hrs after transfection (by Lipofectamine 2000) using the dual luciferase reporter assay system (Promega, Madison, WI). Reporter activity is expressed as the ratio of firefly/Renilla. Assays were done as triplicate transfections, and repeated at least once with similar results. Anchorage-independent growth A pGIPZ shRNAmir construct targeting SMURF1 (V2LHS_229724), along with a non-targeting pGIPZ shRNAmir control, were obtained from Open Biosystems (Huntsville, AL). To create stably-transduced AsPC-1 cell pools, lentiviral constructs (along with Trans-lentiviral packaging mix plasmids) were transfected into 293TN producer cells (System Biosciences, Mountain View, CA), and supernatant packaged virus transduced into AsPC-1 cells following the manufacturer’s instructions (Open Biosystems’ Trans-lentiviral GIPZ packaging protocol). Two days post-infection, 1 mg/ml puromycin (Invitrogen) was added to the culture medium, and cells selected for 14 days. Anchorage independent growth was assayed by colony formation in soft agar. Briefly, 10,000 cells were embedded in a 6-well plate well within a top layer of 0.36% agarose in complete media, over a layer of 0.48% agarose in complete media. Cells were grown for 14–21 days, then visible colonies counted after staining with 0.015% Neutral Red solution. Assays were done as triplicate transductions, and repeated at least once with similar results. NIH-3T3 focus formation assay Full-length human SMURF1 cDNA expression vector, pcDNA3.1-SMURF1 was a kind gift from Di Chen (University of Rochester Medical Center, Rochester, NY), and the parent vector pcDNA3.1 was purchased from Invitrogen (Carlsbad, CA). A catalytic mutant SMURF1 (C699A) [22] was engineered using the QuickChange XL II Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA), with the following mutagenic primers: 59-CGTGGAGGAGACCGCCGGGTTTGCTGTGG -39 (de- PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23924