SMURF1 Amplification in Pancreatic Cancer Figure 4. Analysis of SMURF1 knockdown in other pancreatic cancer lines. (A) Western blot analysis of endogenous SMURF1 protein levels in a panel of pancreatic cancer cell lines. Note that SMURF1 is highly expressed only in the 7q22.1-amplified AsPC-1 cell line. Two different exposures of the SMURF1 blot are shown; GAPDH serves as a loading control. (B) Western blot verification of SMURF1 knockdown (by SMURF1-targeting siRNA pool, compared to non-targeting control siRNA pool) in BxPC-3 and Hs700T cells. (C) Cell proliferation/viability assayed (by WST-1) in BxPC-3 cells following SMURF1 siRNA-mediated knockdown (compared to non-targeting control). Assay done in triplicate (mean +/2 1SD shown); *, P,0.05 (Student’s t-test). (D) Cell proliferation/viability assayed in Hs700T cells, as above. (E) Cell invasion assayed (by Boyden chamber) in BxPC-3 cells following SMURF1 knockdown (compared to non-targeting control). Assay done in triplicate (mean +/2 1SD shown). Note, the reduced proliferation observed with SMURF1 knockdown in Hs700T cells precluded an assay of cell invasion (where invaded cell numbers at 72 hrs are influenced by doubling times). doi:10.1371/journal.pone.0023924.g004 IRB approval) was previously described [6]. Probe labeling and FISH were performed using Vysis (Downers Grove, Illinois) reagents according to the manufacturer’s protocols. A locus- specific BAC mapping to SMURF1 at 7q22.1 (RP11-62N3; BACPAC Resources Centre, Oakland, CA) was labeled with SpectrumOrange, and co-hybridized with SpectrumGreen-la- beled chr 7 centromere probe (CEP7; Vysis). Slides were counterstained with DAPI, and imaged using an Olympus BX51 fluorescence microscope with Applied Imaging (San Jose, CA) Cytovision 3.0 software. Twenty-five tumor cells were scored per case, and amplification defined as an average SMURF1/ CEP7 ratio .2.5. PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23924