NIK regulates PDAC cell proliferation.

<p><b>A:</b> Panc1 or MiaPaca2 cells (5×10⁵ cells, 6cm dish) stably expressing control (scrambled) shRNA or NIK-shRNA (two different sequences, NIK-shRNA1 or NIK-shRNA2) were seeded in E-plates and after attachment cell proliferation was continuously monitored in real-time for indicated times using an xCELLigence RTCA DP instrument. Error bars (gray) represent three experiments. Control analysis of knockdown of NIK for both cell lines is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053676#pone-0053676-g004" target="_blank">Figure 4A</a>. <b>B:</b> Panc1 or MiaPaca2 cells (5×10⁵ cells, 6 cm dishes) were lentivirally infected with control virus or virus for expression of constitutively active NIK (NIK.T559D mutant). The next day media was changed and after 48 hours cells were seeded in E-plates and after attachment cell proliferation was continuously monitored in real-time for indicated times using an xCELLigence RTCA DP instrument. Error bars (gray) represent three experiments. A fraction of the transfected cells were lysed and analyzed for expressed active NIK using Western blot and antibodies directed against FLAG-tagged NIK.T559D (anti-FLAG) or β-actin (anti-β-actin) as a loading control (shown for MiaPaca2). The control blots for the Panc-1 cell line are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053676#pone-0053676-g004" target="_blank"><b>Fig. 4B</b></a>.</p

NIK is a critical regulator of transformed growth in PDAC cells.

<p><b>A:</b> Panc1 or MiaPaca2 cells (5×10⁵ cells, 6 cm dishes) stably expressing control (scrambled) shRNA or NIK-shRNA (two different sequences, NIK-shRNA1 or NIK-shRNA2) were subjected to soft agar colony formation assays. A fraction of the transfected cells were collected and analyzed for NIK expression using Western blot analysis and antibodies directed against NIK (anti-NIK) or β-actin (anti-β-actin) as loading control. The asterisks indicate statistical significance. Scale bars represent 1 mm. <b>B:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were lentivirally infected with control virus or virus for expression of constitutively active NIK (NIK.T559D mutant) and subjected to soft agar colony formation assays. Before seeding a fraction of the cells were lysed and analyzed for expressed active NIK using Western blot and antibodies directed against FLAG-tagged NIK.T559D (anti-FLAG) or β-actin (anti-β-actin) as a loading control. The asterisk indicates statistical significance. Scale bars represent 1 mm. <b>C:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) stably expressing control (scrambled) shRNA, NIK-shRNA1 or NIK-shRNA2 (top row), or cells lentivirally infected with control virus or NIK.T559D mutant (bottom row) were seeded in 3D Matrigel culture. At days 10 (shRNA cells) or 14 (NIK.T559D expressing cells) after seeding colony growth was analyzed by ImagePro. The bar represents 500 µm.</p

Proposed mechanism of NIK/TRAF2 signaling in PDAC cell lines.

<p>Proposed mechanism of how NIK activity is maintained in PDAC cell lines. In normal pancreatic ductal cells TRAF2 is expressed and forms a degradation complex for NIK with TRAF3, cIAP1/2. This leads to ubiquitination and degradation of NIK. In PDAC cells, TRAF2 is degraded by ubiquitination. This prevents formation of a NIK degradation complex and NIK remains expressed. NIK signals to the non-canonical IKK complex and IKKα mediates activation of NF-κB2 and formation of RelB/p52 dimers. Net effect of such signaling is an increase in pancreatic cancer cell proliferation.</p

TRAF2 expression is downregulated in PDAC cell lines.

<p><b>A:</b> Cell lysates of indicated cells were normalized to 0.5 mg/ml and then 20 µg were subjected to SDS-PAGE. Samples were transferred to nitrocellulose and analyzed by Western blot for expression of TRAF2 (anti-TRAF2), TRAF3 (anti-TRAF3), cIAP1 (anti-cIAP1), cIAP2 (anti-cIAP2) or β-actin (anti-β-actin; loading control). TRAF2 overexpressed in Hek293 cells served as an additional molecular weight control. <b>B:</b> Samples of mRNA of indicated cell lines were subjected to quantitative RT-PCR directed against TRAF2. Samples were normalized to GAPDH. <b>C:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were co-transfected with TRAF2 and vector control or HA-ubiquitin. After 24 hours cells were lysed, TRAF2 immunoprecipitated (anti-TRAF2) and analyzed by immunoblotting for ubiquitination of TRAF2 (anti-HA). Blots were re-probed for TRAF2 (anti-TRAF2). <b>D:</b> Indicated cell lines were treated with MG-132 (20 µM) for 0, 4, 8, 16 or 24 hours. Cells were lysed and analyzed for expression of endogenous TRAF2 (anti-TRAF2) or β-actin (anti-β-actin; loading control. TRAF2 overexpressed in Hek293 cells served as positive control. <b>E:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were transfected with vector control or TRAF2 as indicated. After 24 hours cell lysates were analyzed by Western blotting for expression of NIK (anti-NIK), overexpressed TRAF2 (anti-TRAF2) or β-actin (anti-β-actin) as loading control.</p

TRAF2 and NF-κB2 regulate PDAC cell proliferation.

<p><b>A–C:</b> Indicated cell lines (5×10⁵ cells, 6 cm dishes) were infected with control or TRAF2 adenovirus. After 48 hours cells were seeded in E-Plates and after attachment cell proliferation was continuously monitored in real-time for 30 hours using an xCELLigence RTCA DP instrument. Error bars (gray) represent three experiments. A fraction of the transfected cells were lysed and analyzed by Western blot for TRAF2 or β-actin (loading control). <b>D:</b> HPDE cells (5×10⁵ cells, 6 cm dishes) were infected with control or NF-κB2/p100 adenovirus. After 48 hours cells were seeded in E-Plates and after attachment cell proliferation was continuously monitored in real-time for 20 hours using an xCELLigence RTCA DP instrument. Error bars (gray) represent three experiments. A fraction of the transfected cells were lysed and analyzed by Western blot for processed, active NF-κB2 using antibodies directed against p52 (anti-p52) or β-actin (anti-β-actin) as a loading control.</p

Expression of TRAF2, NIK and NIK activity in human samples for pancreatic cancer.

<p><b>A:</b> Tissue microarrays (TMAs) including 10 normal pancreatic tissue samples and 55 pancreatic ductal adenocarinoma were H&E stained or analyzed for the expression of TRAF2 (anti-TRAF2), NIK (anti-NIK) or active NIK (anti-pT559-NIK). Representative pictures of tumor tissues are depicted. Numbers indicate the position of the tissue on the TMA. The bar indicates 50 µm. <b>B:</b> Analysis of correlation of TRAF2, NIK, and pT559-NIK in n = 55 human samples for pancreatic addnocarcinoma. Top pie graph shows percentage of cells with low TRAF2 expression and high expression of NIK and pT559-NIK in red, percentage of cells with high TRAF2 and NIK expression and low expression of pT559-NIK in green, and percentage of cells with high TRAF2, NIK and pT559-NIK expression in blue. Bottom bar graphs show percentage of these groups in grade1, grade 2, and grade 3 tumors.</p

Active PKD1 directly phosphorylates SNAI1 at S11.

<p><b>A:</b> The amino-acids surrounding serine 11 in SNAI1 form a PKD consensus motif as it was described for S82 of Hsp27 and S978 of SSH1L. <b>B:</b> PKD phosphorylates SNAI1 at S11 in an <i>in vitro</i> assay. Bacterially-expressed and purified GST (negative control), GST-SNAI1 or GST-SNAI1.S11A were incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the pMOTIF antibody, which recognizes the phosphorylated PKD motif in PKD substrates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030459#pone.0030459-Doppler1" target="_blank">[45]</a> or with the novel pS11-SNAI1 antibody specifically generated for this site. Control blots were performed for protein input (anti-PKD1, anti-GST). <b>C, D:</b> HeLa cells were transfected with combinations of vector control, active PKD1 (PKD1.CA) and SNAI1 or SNAI1.S11A mutant as indicated. PKD-mediated phosphorylation of SNAI1 was detected using the pMOTIF (C) or the pS11-SNAI1 (D) antibodies. <b>E, F:</b> HeLa cells were transfected with combinations of vector control, active RhoA (RhoA.CA) and PKD1 or PKD1.KD mutant (E) or control shRNA and shRNA specific for PKD1/2 (F) as indicated and FLAG-tagged SNAI1. PKD-mediated phosphorylation of SNAI1 was detected using the pS11-SNAI1 antibody. Samples were also control-stained for SNAI1 and PKD1 expression using anti-FLAG or anti-PKD1 antibodies, respectively. Anti-GST control staining for RhoA.CA and GST control are depicted in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030459#pone.0030459.s002" target="_blank">Figure S2</a></b>. <b>G:</b> NMuMG cells were treated with TGFβ1 (10 ng/ml) for 48 hours. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or PKD1 activity (anti-pS738/742-PKD) or total PKD1 expression (anti-PKD1) as indicated. <b>H:</b> NMuMG cells were treated with CID755673 (25 µM, 4 hr) or left untreated as indicated. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or SNAI1 expression (anti-SNAI1).</p

PKD regulates E-cadherin expression in epithelial cells.

<p><b>A:</b> NMuMG cells were transfected with GFP-tagged, kinase-dead PKD1 (PKD1.KD) and endogenous expression of E-cadherin was determined with immunofluorescence staining (anti-E-cadherin). DAPI staining served as a nuclear marker (bar is 50 µm). <b>B:</b> MCF-7 cells were transfected with vector control, HA-tagged PKD1 or kinase-dead PKD1 (PKD1.KD). After 48 hours, samples were analyzed by Western blot for E-cadherin expression (anti-E-cadherin) as well as expression of PKD1 (anti-PKD1). Staining for actin (anti-actin) served as loading control. <b>C:</b> MCF-7 cells were transfected with vector control, HA-tagged constitutively-active PKD1 (PKD1.CA) or kinase-dead PKD1 (PKD1.KD) as well as E-cadherin promoter luciferase gene reporter and renilla luciferase reporter. Induced luciferase activity was measured. Error bars shown represent standard deviations. The asterisks indicate statistical significance (p<0.05) as compared to vector control.</p

Phosphorylation of SNAI1 decreases its binding to Ajuba.

<p><b>A:</b> HeLa cells were co-transfected with MYC-tagged Ajuba and vector control, and FLAG-tagged wildtype SNAI1, SNAI1.S11A or SNAI1.S11E mutants as indicated. Ajuba was immunoprecipitated (anti-MYC) and precipitates were analyzed for co-precipitated SNAI1 (anti-FLAG). Samples were re-stained for Ajuba (anti-MYC) and lysates were control-stained for expressed SNAI1 (anti-FLAG). <b>B:</b> Proposed mechanism of how PKD1-mediated phosphorylation regulates SNAI1 function as a transcriptional repressor of E-cadherin gene expression.</p

PKD1 conserves the epithelial phenotype in normal mammary gland cells.

<p><b>A:</b> NMuMG cells were either left untreated or were treated with TGFβ1 (10 ng/ml) for 48 hours. Cell morphology was photographed (bar is 200 µm) and cells were harvested and analyzed for expression of epithelial (E-cadherin, cytokeratin) and mesenchymal (N-cadherin) markers by Western blotting with anti-N-cadherin, anti-E-cadherin, or anti-cytokeratin antibodies. Staining for actin (anti-actin) served as a loading control. <b>B:</b> NMuMG cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Endogenous PKD1 was immunoprecipitated (anti-PKD1) and analyzed for phosphorylation at its activation loop that correlates with its activity (anti-pS738/742-PKD), or samples were control stained for total PKD1 (anti-PKD1). <b>C:</b> Cells were stimulated with PMA (100 nM, 10 min), EGF (50 ng/ml, 10 min), Bradykinin (0.5 µg/ml, 10 min) or left untreated. Endogenous PKD1 was immunoprecipitated and subjected to an <i>in vitro</i> kinase assay using PKD substrate peptide. PKD1 activity is depicted relative to PMA-activated PKD1 (maximum activation). Equal immunoprecipitation was controlled by SDS-PAGE and immunoblot (anti-PKD1). <b>D:</b> NMuMG cells were either transfected with control vector or with active PKD1 (PKD1.CA, PKD1.S738E.S742E). 24 hours after transfection, cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Lysates were analyzed for expression of N-cadherin, E-cadherin, expression of PKD1, or actin as a loading control. <b>E:</b> NMuMG cells were stably-transfected with vector control, wildtype PKD1 or kinase-dead PKD1.K612W (PKD1.KD) Cell morphology was analyzed by brightfield microscopy (bar is 200 µm). Expression of endogenous and overexpressed PKD1 was determined by Western blot analysis using an anti-PKD1 antibody. Immunoblotting for actin (anti-actin) served as loading control.</p