SDS-PAGE, and Ubc13 and Uev1 detected by Western blotting with specific antibodies [61]. Surface plasmon resonance Recombinant Uev1 protein was immobilized on a Biacore CM5 Chip. The final response obtained was 1179,1 RU (response units) 2 (1 response unit corresponds to 1 pg of protein per mm ) of immobilized Uev1. Purified Ubc13 was maintained at 400 nM in running buffer (10 mM HEPES, 0.15 M NaCl, pH 7.4). Ubc13 was preincubated with the test compounds at varying concentra- tions, and interaction assays run in a Biacore T100 instrument. In vitro ubiquitylation Purified Ubc13 (0.2 mM), Uev1 (0.2 mM), and E1 (0.1 mM, Boston Biochem) were incubated in 50 mM Tris-HCl, 5 mM MgCl2, 2 mM ATP and 0.5 mM DTT, with either 117 mM wild- type ubiquitin (Biomol), 6 mM UbK48_only or 6 mM Ub- K63_only (Boston Biochem). To determine the effects of compounds Ia and IIa, Ubc13 was pre-incubated for 10 min at room temperature with 1–100 mM of either compound. The reactions were allowed to proceed for the indicated times and stopped by addition of SDS-PAGE sample buffer and boiling. The reaction products were detected by Western blotting with a rabbit anti-ubiquitin antibody (Biomol). Cell culture HeLa and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). PC-3/M cells [62] were kindly provided by Dr. Isaiah Findler, and PC-3/luc cells were described in [42]. All cells were grown in an atmosphere of 5% CO2, 95% humidity at 37uC in RPMI 1640 or DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine and 0.1 mM non-essential aminoacids (PAA, Pasching, Austria). PC-3/luc cells were grown in complete medium supplemented with 200 mg/mL Geneticin (Sigma, Alcobendas, Spain) to maintain integrated copies of the luciferase gene. Chemosensitization assays in mammalian cells HeLa and PC-3 cells were treated with varying concentrations of doxorubicin or etoposide, together, or not, with 100 mM of compound Ia. Viable cell numbers were determined at 24, 48 and 72 hours with the CyQuant assay. In vivo ubiquitylation of PCNA HEK293T cells, transfected with pcDNA-HA-UbK63, were incubated overnight with 100 mM of compound Ia, followed by UV irradiation (60 J/m2). After 6 h, cells were harvested, immunoprecipitated with anti-PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), immune complexes resolved by SDS-PAGE and immunoblotted with anti-HA rat monoclonal antibody (Roche, Mannheim, Germany). As a loading control, immunoprecipitates were immunoblotted with anti-PCNA. NF-kB assays To determine IkB protein levels, HeLa cells were incubated overnight with compound Ia, and treated with TNF-a (20 ng/mL) for 15 min. Cells were lysed and analyzed by Western blotting with anti-IkB (Cell Signaling, Danvers, MA). To analyze IKKa/b phosphorylation, the same lysates as above were tested by Western blotting with an antibody to phospho-IKKa (Ser180)/b (Ser181), or to total IKKa (Cell Signaling). For nuclear translocation assays, Novel Ubc13 Inhibitors HeLa cells grown on sterile coverslips were transfected with pPCR3/p65-GFP, incubated overnight with varying concentra- tions of compound Ia, and treated with TNF-a (20 ng/mL) for 30 min. Samples were fixed, counterstained with Hoechst 33258, and images captured with a fluorescence microscope. At least 3 coverslips and 200 cells from 10 separate fields were counted and scored for positive nuclear green flourescence. The proportion of cells with nuclear p65-GFP in untreated cells was taken as background. Background subtracted values were normalized against those for cells treated with TNF-a alone. Invasiveness assays PC-3/luc cells (56104), preincubated for 24 h with compound Ia, were resuspended in 0.1% BSA and seeded on 12-well, 8-mM pore diameter Transwell chambers (Costar, Cambridge, MA) coated with diluted (1:20 in H2O) growth factor-reduced Matrigel (Beckton-Dickinson, San Jose, CA). After 24 h of incubation in the presence of compound Ia in both chambers, cells in the top chamber were removed, cells in the lower chamber were assayed for luciferase activity, and cell numbers inferred from standard luciferase-activity curves. Each condition was assayed in triplicate. Soft agar colony formation Melted agar (0.5%) in complete medium was placed at the bottom of 12-well plates, allowed to solidify and overlayed with 36103 PC-3/M cells resuspended in 0.3% agar/complete medium. Cells were fed three times a week with medium containing compound Ia. After 2 weeks, wells were fixed with 0.5% glutaraldehyde, stained with 0.025% crystal violet, images captured and analyzed with the Image J software. Only colonies $0.1 mm diameter were scored. Each condition was assayed in triplicate. Tumor xenograft analyses PC-3/luc cells (56105) were injected into each rear limb of male Balb/c nu/nu mice. Tumor size was assessed by photon counts in a ORCA-2BT instrument (Hamamatsu Photonics, Hamamatsu City, Japan). Luciferin (16.7 mg/ml in PBS) (Promega) was injected intraperitoneally into anestesized mice, and images acquired for 5 min and quantitated. Light measurements were expressed as photon counts (PHCs), and values normalized vs. initial counts for each tumor. Supporting Information Supporting Methods S1 Found at: doi:10.1371/journal.pone.0011403.s001 (0.07 DOC) Table S1 Found at: doi:10.1371/journal.pone.0011403.s002 (0.03 PDF) Table S2 Found at: doi:10.1371/journal.pone.0011403.s003 (0.03 PDF) MB MB MB Figure S1 Antagonism by compounds Ia and IIa of the Ubc13- Uev1 interaction, assayed by yeast two-hybrid. Yeast cells (AH109 strain), co-transfected with pBD-Ubc13 and pACT2-Uev1 and grown in quadruple selection medium, were incubated overnight with 100 mM of cyclic compounds Ia or IIa, and assayed for a- galactosidase activity as a semiquantitative measure of strength of interaction. Values were normalized against those of cells incubated with a control, unrelated cyclic compound. As an PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11403