cDNA array production and hybridization

Production of cDNA arrays, extraction and radioactive labeling of total RNA, as well as hybridization and quantification of hybridization signals were performed as previously described [24,25]. In brief, cDNAs were PCR amplified and arrayed in duplicate onto nylon membranes using robotic devices.

Total RNA reverse transcribed and radiactively labeled with 33P-dATP using the StripEZ-RT Kit (Ambion) and hybridized over night to nylon membrane arrays in ULTRArray hybridization buffer (Ambion) at 50°C. Radioactive signals were detected using a STORM phosphor imaging system (Amersham Biosciences, Feiburg, Germany) and quantified with the ArrayVision software (InterFocus, Haverhill, Great Britain). Signal intensities were normalized to the mean signal intensity of all features on an individual array.

Genes were defined as differentially expressed between two sets of samples if: (1) the mean normalised expression value exceeded 0.5 in at least one of the two sample sets, (2) the difference between the mean normalised expression values was at least twofold between the sample sets and (3) a two-sided t test yielded a p value of less than 0.05.

Raw data of all experiments can be accessed at http://www.staff.uni-marburg.de/~buchhol3/RevTrans/

The following gene expression profiling experiments of tissues and in vivo/in vitro models for serial characterization of candidate genes were conducted:

Cell lines for parallelized and individual functional assays

Panc-1 [31] and HEK-293 [32] cells were obtained from the American Type Culture Collection (Manassas, USA). S2-007 and S2-028 were from T. Iwamura [33] (Miyazaki Medical College, Miyazaki, Japan). IMIM-PC1 cells were kindly provided by F.X. Real [34] (CNIO, Madrid, Spain).

Cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% FBS and 0.05 mg/ml Gentamicin at 37°C and 5% (v/v) CO2.

Reverse transfection arrays

Full length open reading frames (ORFs) of all candidate and control genes were purchased as cDNA clones from Open Biosystems (Lafayette, CO, USA), PCR-amplified using specific primers and cloned into the pENTR vector (Invitrogen/Life Technologies, Darmstadt, Germany). ORFs were then shuttled into the pdECFP and pdEYFP expression vectors [35] using the Gateway cloning system (Invitrogen/Life Technologies, Darmstadt, Germany). Each ORF was therefore available as an N-terminal fusion construct with cyan fluorescent protein (CFP) as well as a C-terminal fusion construct with yellow fluorescent protein (YFP).

For production of ‘reverse transfection microarrays’, expression constructs were spotted onto glass slides according to a protocol modified from [22]. 4 μg of each plasmid DNA was mixed with 11 μl PBS and 2 μl Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) in a 96-well cell culture plate. After 20 min incubation at RT, 15 μl gelatine (0.1% in PBS) was added to each well. Transfection mixtures were arrayed on Superfrost Plus glass slides (Menzel GmbH, Braunschweig, Germany) using a Tecan Miniprep 75 automated sample processor (Tecan, Männedorf, Switzerland), generating spot sizes of 0.5–1.5 mm diameter. Slides were dried and stored up to 3 weeks in a dry atmosphere at 4°C. For generation of live cell microarrays, slides were placed in quadriPERM plates (Sarstedt, Nümbrecht, Germany), seeded with 4.5 x 10⁵ HEK-293, 4 x 10⁵ Panc-1, or S2-007: 5 x 10⁴ S2-007 in DMEM/10% FCS and incubated for 48 h at 37°C.

Immunocytochemistry

Live cell microarrays were fixed for 15–30 min at room temperature with 4% paraformaldehyde solution in PBS and counterstained and mounted using DAPI-containing mounting medium (Vector Laboratories, Burlingame, USA). Expression of fusion constructs and subcellular localization of the gene products were documented using a Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss GmbH, Göttingen, Germany). Where applicable, optical sections of fluorescent samples were produced at 630x magnification using the Zeiss ApoTome technology (http://www.zeiss.com/microscopy/en_de/products/imaging-systems/apotome-2-for-biology.html).

Antibody incubation and detection were carried out according to the manufacturer’s instructions for the individual antibodies, respectively. The following antibodies were used:

1. anti-Ki67: 1:400, polyclonal rabbit, ab833, Abcam, Cambridge, UK
2. anti-Cyclin B1: 1:100, monoclonal mouse, cat. #4135, Cell Signaling Technology, Danvers, USA
3. anti-ACTIVE-Casapse-3: 1:150, polyclonal rabbit, cat. #G7481, Promega, Madison, USA
4. anti-Vimentin: 1:200, monoclonal mouse, ab28028, Abcam, Cambridge, UK
5. anti-E-Cadherin: 1:100, monoclonal mouse, cat. #610181, BD Biosciences, Sparks, USA
6. anti-rabbit IgG (Cy3) 1:500, polyclonal goat, ab6939, Abcam, Cambridge, UK
7. anti-mouse IgG (Cy3) 1:500, polyclonal goat, ab97035, Abcam, Cambridge, UK

mRNA isolation and protein extraction

RNA isolation was carried out using the peqGOLD Total RNA Kit (peqlab Biotechnology GmbH, Erlangen, Germany) according to the manufacturer’s instructions. Concentrations were quantified with the NanoDrop ND-1000 (peqlab Biotechnology GmbH) followed by synthesis of first-strand cDNA from 1 μg total RNA using the Omniscript Kit and protocol (Qiagen, Hilden, Germany).

For total protein extracts, cells were centrifuged in culture medium (13 000 rpm, RT, 5 min). Pellets were resuspended in PBS, supplemented with proteinase inhibitor (G-Biosciences, Maryland Heights, USA) and sonicated with a Labsonic U (B.Braun, Melsungen, Germany). Protein concentrations were determined using Bradford protein assay and measured with the Multiskan FC photometer (Thermo Scientific, Langenselbold, Germany).

qRT-PCR and immunoblotting

First-strand cDNAs were amplified in 7500 Fast Real-Time PCR System (Applied Biosystems, Warrington, USA) using Power SYBR Green Master Mix (Applied Biosystems) and specific primer pairs designed with the PrimerExpress program (Applied Biosystems, for sequences see supplementary material).

For Western blot, 10 ng of total protein extracts were loaded and separated by 10 or 15% SDS-PAGE. Separated proteins were transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany) and blocked for 4–6 h at 4°C in 1xTBS, 0.1% Tween 20 and 5% powdered milk. Primary antibodies were diluted 1:1 000 in blocking buffer and incubated overnight at 4°C. TBS-T (0.1%) was used as washing buffer. The HRP-conjugated goat-anti-rabbit secondary antibody was diluted to 1:10 000 and incubated for 1–2 h at 4°C in blocking buffer. Proteins were detected using the ECL immunoblot kit (GE Healthcare Europe GmbH, Freiburg, Germany).

Construction and analysis of tissue microarrays

For the construction of tissue microarrays (TMAs), 1 mm (tumor) and 1.5 mm (normal tissue) sized tissue biopsies were extracted from paraffin donor blocks and transferred into pre-punched holes on recipient paraffin blocks with a tissue microarrayer (Beecher Instruments, Inc., Sun Prairie, USA) equipped with a TMA booster (Alphelys, Plaisir, France). Grid layouts for tumor and normal pancreatic tissue TMAs were designed with the TMA Designer 2 software (Alphelys). The recipient blocks were sealed for 10 min at 56°C and 30 min at 4°C. This procedure was repeated twice. The TMA blocks were cut into 3.5 μm sections and placed on SuperFrost Plus slides for immunohistochemical staining. The use of human tissues for research purpose was approved by local ethics committee at the University Hospital, Tübingen (470/201BO1), and Clinic of Surgery, University Heidelberg, Germany (301/2001).

Immunohistochemical staining was performed with a mouse monoclonal anti-ADRBK1 antibody (Thermo Fischer, #MA5-15840; 1:1000 dilution) on the Ventana BenchMark XT system (Ventana Medical Systems, Tucson, USA) including the deparaffinization of the formalin-fixed paraffin embedded tissue sections and the heat-induced antigen-retrieval.

Analysis of staining intensities was performed by a pathologists with expertise in pancreatic tumors (B S) in a blinded fashion regarding tumor parameters. The proportion of the positive cells showing a significant staining in tumor areas was estimated in percent and divided into scores (1–10%-weak, 11–50%-moderate, > 50%-strong.

Transfection of siRNAs

For RNAi-mediated silencing of ADRBK1 expression, pre-designed Silencer siRNAs (Applied Biosystems/Ambion, Austin, TX, USA) were transfected in a concentration of 40 nmol using the siLentFect Lipid Reagent (Bio-Rad, München, Germany). Accell non-targeting siRNA#1 (Thermo Fisher Scientific, Dharmacon Products, Lafayette, LA, USA) was employed as non-silencing control.

Proliferation and viability assays

For BrdU incorporation assays using the Cell Proliferation ELISA Kit (Roche Diagnostics, Mannheim, Germany), 5 000 to 7 500 cells were reseeded 24 h post-transfection into a ViewPlate-96 Black cell culture plate (Perkin Elmer, Waltham, MA, USA). Following over-night culturing, cells were incubated 4–6 h with BrdU containing medium. After fixation for 1 h, cells were stained for 1.5 h with the peroxidase-conjugated anti-BrdU antibody, chemiluminescence substrate added and the emitted light quantified with a Centro LB 960 luminometer (Berthold Technologies GmbH, Stuttgart, Germany).

For MTT assays, 20 000 to 25 000 cells were reseeded 24 h after transfection in 24-well cell culture plates. After additional culturing for 24 h or 48 h, supernatant was replaced by MTT-containing medium (thiazolyl blue, Carl Roth GmbH) and plates were incubated for 2 h at 37°C. Medium was replaced by solubilization solution (10% Triton-100, 0.1 M hydrochloric acid in 2-propanol). Extinction was measured at 570 nm with the Multiskan FC photometer (Thermo Scientific, Schwerte, Germany).

Flow cytometry

Trypsinized cells were centrifuged at 1,200 rpm for 3 min. After washing with PBS, pellets were thoroughly resuspended in 50–100 μl PBS and the cell suspension transferred drop-wise into ice-cold ethanol (70%) under continuous vortexing. Following centrifugation at 1 000 g for 5 min, the supernatant was carefully discarded, the pellet washed with 500 μl ice-cold PBS and resuspended in 500 μl of propidium-iodide mixture (1 mg/ml Propidium-iodide (Sigma Aldrich) + 500 μg/ml DNAse-free RNAse (Roche Diagnostics) in PBS). Samples were incubated for 30–45 min at RT in the dark and subsequently measured with the flowcytometer LSR II (BD Biosciences, Heidelberg, Germany). For data evaluation, the software ModFit LT (Verity Software House, Topsham, USA) was used.

Serial characterization of PDAC candidate genes by expression profiling using cDNA array hybridizations, data analysis and selection of candidate genes

As a first step in the systematic identification of novel functionally relevant candidate genes in pancreatic cancer, we produced custom cDNA arrays with pancreatic cancer-specific cDNA clone collections (n = 468 from [7] and [36]) which were additionally augmented with large-scale collections of cDNA clones (n = 1492) from potentially relevant gene families such as kinases, phosphatases, serum response genes etc. (see data sets in supplementary information for details of the array compositions). These arrays were then used for gene expression analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of pancreas development, cell differentiation, invasion, metastasis, apoptosis resistance etc. (note that since the cDNA clone collections for the generation of the arrays was continually expanded over the course of these studies, not all cDNAs were hybridized with all experimental models). In order to be selected for further functional characterization, candidate genes had to be significantly deregulated in pancreatic cancer (overexpressed or downregulated in primary pancreatic cancer tissues as compared to chronic pancreatitis and normal pancreas samples), as well as show significant regulation in at least one of the functional model systems (see Material&Methods section for details of hybridization signal quantification and definition of differential expression).

This raw list of potential candidate genes was manually curated to exclude genes which had previously been intensively studied in pancreatic cancer, as well as exclude expressed sequence tags (ESTs) from the pancreatic cancer-specific cDNA collection which showed insufficient homology to annotated human genes. Using these criteria, a total of 50 genes were selected for parallelized functional characterization (see below). This list was augmented with 17 candidate genes which had shown PanIN-specific expression patterns in our previous analyses of microdissected samples of normal ducts as well as pre-cancerous and cancerous lesions from human pancreata [11]. Finally, a selection of 14 known genes with diverse cellular functions were added as positive controls for the functional analyses. The complete list of selected candidate and control genes is given in S1 Table (supplementary information).

Parallelized cell-based assays

In order to rapidly screen for functional effects of all 67 candidate genes in parallel, expression constructs of the candidate genes as well as 14 control genes with known functions were cloned in the form of N-terminal fusions with cyan fluorescent protein (CFP) as well as C-terminal fusions with yellow fluorescent protein (YFP). The plasmid DNA, complexed with transfection reagents, was arrayed onto standard microscopy glass slides and overlaid with suspensions of pancreatic cancer cells (Panc-1, S2-007) or non-transformed control cells (HEK-293). Under a fluorescence microscope, spots of transfected cells were easily identified due to the specific fluorescent signals of the CFP or YFP fusion proteins, which also allowed direct comparison with neighbouring non-transfected cells.

The reverse transfection arrays were then utilized for parallelized functional analyses in order to identify novel candidate genes with potentially important functions in pancreatic cancer cells. In a first round of evaluation, subcellular localization of the gene products (as well as overall morphology compared to non-transfected cells), both in the presence and absence of serum, was recorded and compared between cancer and control cells (see Fig 2 for examples of different localizations). In subsequent analyses, arrays of fixed cells were incubated with antibodies against well-defined markers of apoptosis (cleaved caspase-3), proliferation (Ki-67, Cyclin B) and epithelial-mesenchymal transdifferentiation (EMT; mesenchymal marker vimentin, epithelial marker E-cadherin). Examples of staining results are displayed in Fig 3. Frequency and intensity of staining were recorded, visually scored and compared between transfected and non-transfected cells. Recurring differences between transfected and non-transfected cells were graded as “++” (strong effects), “+” (moderate but reproducible effects) and “(+)” (weak and/or variable effects). Table 1 lists all genes for which at least one score of “++” or at least three scores of “+” were recorded in independent replicate experiments and which were thus defined as suitable for individual in-depth analyses. The selected candidates comprised 3 kinases (ADRBK1, FASTK, PRKCZ), 3 phosphatases (PPM1G, PPP2R1A, PPP2R4), 2 transcription factors (FRA-2, GTF2F2), 2 actin-interacting proteins (CFL1, RRAS), 1 transmembrane protein (TM4SF1), 1 histone methyl transferase (SUV39H1), 1 G protein-coupled receptor (EBI2) and 1 interferone-inducible protein with unknown cellular function (IFI27). None of them had been associated with pancreatic cancer at the time they were included in the reverse transfection arrays.

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Fig 2. Examples of distinct subcellular localization of fluorescence-tagged candidate gene peoducts after reverse transfection.

Shown are CFP (A) or YFP (B-C) fusion constructs. Following transfection and fixation of cells on reverse transfection microarrays, coverslips were applied using DAPI-containing mounting medium. For CFP fusion proteins, DAPI signals are shown in brown (false color) to provide suitable contrast (A). Shown are examples of purely cytoplasmic (A), uniform nuclear-cytoplasmic (B) and purely nuclear localization (C) as well as localization to endosome-like structures (D). Original magnifications are indicated in the images.

https://doi.org/10.1371/journal.pone.0122946.g002

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Fig 3. Examples of the different antibody stainings for functional analyses.

A: Caspase-3 staining (Cy-3, red), HEK-293 cells transfected with H1F0-YFP (green); B: Ki-67 staining (Cy-3, red), HEK-293 transfected with NKX2-5-YFP (green); C: Cyclin B staining (Cy-3, red), PANC-1 transfected with PRKCZ-YFP (green); D: Vimentin staining (Cy-3, red), PANC-1 transfected with FASTK-YFP (green); E: E-Cadherin staining (Cy-3, red), PANC-1 transfected with PPP2R1A-YFP (green). Original magnifications are indicated in the images.

https://doi.org/10.1371/journal.pone.0122946.g003

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Table 1. List of genes showing reproducible effects in parallelized assays.

https://doi.org/10.1371/journal.pone.0122946.t001

Functional validation / in-depth individual characterization of candidate gene ADRBK1

As a proof-of-concept to confirm the validity of functional data obtained via the parallelized cell-based assays, we exemplarily show detailed in vitro functional characterization data of one gene, ADRBK1, which was the first gene (in alphabetical order) on the list of high-priority candidates (Table 1). Detailed in vivo and in vitro characterization of other candidate genes up to the generation of genetic engineered mouse models are either in press or in preparation and will be reported elsewhere.

Subcellular localization of the ADRBK1/CRK2 protein.

ADRBK1 (Adrenergic, Beta, Receptor Kinase 1) also known as GRK2 (G-Protein Coupled Receptor Kinase 2) was originally included in the cDNA arrays as a member of the kinase gene family, which represent ideal druggable target genes. It was found to be overexpressed in PDAC during the serial expression profiling experiments. In the reverse transfection array experiments, ADRBK1/CRK2 had shown marked differences in subcellular localization depending on the presence or absence of serum (cytoplasmic localization in the presence, nuclear and cytoplasmic localization in the absence of serum). Traditional (“forward”) transfection of ADRBK1 fusion constructs in Panc1 cells confirmed this observation, showing the expected fluorescent signals in the nucleus exclusively in serum-deprived cells 24h after transfection (Fig 4A and 4B). Interestingly, after prolonged incubation times, there was a strong tendency of the signal to shift towards the nucleus regardless of the presence or absence of serum (Fig 4C and 4D), possibly indicating a role of ADRBK1 in the cells’ reaction to the depletion of nutrients and/or growth factors.

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Fig 4. Validation of growth condition-associated subcellular localization of ADRBK1.

ADRBK1-YFP fusion constructs (green signals) were transfected into cultured Panc-1 cells cultured in the absence (A, C) or presence (B, D) of 10% serum. After 24h or 48h of incubation, cells were fixed and counterstained with DAPI (blue signals). After 24h, ADRBK1-YFP signals were detectable in both, cytoplasm and nuclei, of cells cultured without serum (A), while nuclei of cells cultured with serum were devoid of YFP fluorescence (B). In contrast, after 48h of incubation, cells grown under either condition showed prominent staining of nuclei (C, D). Optical sections of fluorescent images were obtained using the ApoTome technology. Original magnifications are indicated in the images.

https://doi.org/10.1371/journal.pone.0122946.g004

ADRBK1 overexpression promotes growth in human PDAC.

In order to confirm that ADRBK1 is overexpressed in human pancreatic cancer, primary human tissues were analyzed for ADRBK1 expression by quantitative RealTime PCR. ADRBK1 mRNA levels were undetectable in all normal pancreas samples analyzed (n = 8), as well as in 4 of 5 chronic pancreatitis samples, whereas strong expression was detected in 5 out of 10 pancreatic cancer tissues (Fig 5A). More detailed analyses on the protein level corroborated these results. Immunohistochemical staining of tissue microarrays (TMAs) comprising 33 normal pancreas samples, 28 samples of chronic pancreatitis tissues and 65 PDAC samples (grades 1–3) cornfirmed that ADRBK1 protein was undetectable in all non-neoplastic tissues (chronic pancreatitis and normal pancreas), whereas ADRBK1 staining was readily detectable in 33 out of 65 (51%) PDAC samples (Fig 5B). Interestingly, ADRBK1 signal in positive samples was detectable in both, epithelial cancer cells as well as subsets of infiltrating immune cells (Fig 5B, upper right panel). Staining frequency and/or intensity did not vary systematicall with tumor grade (Fig 5B, lower panel).

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Fig 5. ADRBK1 overexpression promotes growth in human PDAC.

A: Box-and-whisker plot showing ADRBK1 mRNA expression in primary human pancreatic tumor tissue samples, chronic pancreatitis and normal pancreas as analyzed by quantitative realtime reverse transcription PCR (qRT-PCR). Expression was normalized to ribosomal protein, large, P0 (RPLP0) mRNA levels. Data in Fig. represent median and 2nd and 3rd quartiles (boxes) as well as minimum and maximum values (whiskers). CP = Chronic Pancreatitis. ** p<0.01, *** p<0.001 (Student’s t-test). B: Immunohistological analyses of ADRBK1 expression using human pancreatic tissue microarrays (TMAs) comprising 33 donor, 28 chronic pancreatitis and 65 tumor tissues. Micrographs are representative of typical staining results for donor samples (left panel), which were devoid of ADRBK1 staining, or ADRBK1-positive neoplastic tissues (right panel) (original magnification 100x). Note that in positive PDAC cases, neoplastic ducts as well as individual infiltrating immune cells (arrows) stained postitve for ADRBK1. Lower panel: Distribution of staining intensities across different tumor grades (G1: n = 23; G2: n = 19; G3: n = 23). C: TTK mRNA expression levels in a variety of different pancreatic cancer and control cell lines (qRT-PCR; expression was normalized to ribosomal protein, large, P0 (RPLP0) mRNA levels). D, E: MTT assays demonstrated increased numbers of viable S2-007 (C) and Panc-1 (D) cells 72h after transfection of fluorescence-tagged ADRBK1 expression constructs (CFP: N-terminal CFP-ADRBK1 fusion; YFP: C-terminal ADRBK1-YFP fusion) compared to GFP controls in the presence (dark bars) or absence (light bars) of 10% serum in the culture medium.

https://doi.org/10.1371/journal.pone.0122946.g005

Analysis of ADRBK1 mRNA levels in a broad panel of transformed as well as non-transformed cell lines revealed profound differences in ADRBK1 expression. Of interest, Panc-1 and S2-007 cells, which had been used for the parallelized functional assays with recombinant expression of fluorescence-tagged candidate genes, showed no detectable endogeneous ADRBK1 expression, while the majority of pancreatic cancer cell lines expressed moderate or high levels of ADRBK1 mRNA (Fig 5C). In order to assess the functional consequences of recombinant ADRBK1 expression in the negative cell lines, S2-007 and Panc-1 cells were transiently transfected with CFP-ADRBK1 or ADRBK1-YFP expression constructs or GFP as a negative control, respectively. MTT cell viability assays performed 72h after transfection demonstrated a trend to increased proliferation in the ADRBK1-expressing cells compared to GFP controls, although statistical significance was only reached in the case of S2-007 cells transfected with the CFP-ADRBK1 construct (Fig 5D and 5E).

Inhibition of endogeneous ADRBK1 expression strongly impairs cell growth.

To more thoroughly study the role of endogeneous ADRBK1 in pancreatic cancer cells, two cell lines with moderate (PaTu 8988t) and high (S2-028) mRNA expression were subjected to siRNA-mediated transient inhibition of ADRBK1 expression. As an additional reference, immortalized but non-transformed HEK293 cells, which also show strong ADRBK1 mRNA expression, were likewise examined. siRNA transfection routinely resulted in ~80% reduction in ADRBK1 mRNA levels in all three cell lines and with all three independent siRNAs (Fig 6A). While MTT assays uniformly demonstrated a clear and statistically significant reduction in the numbers of viable cells for all three cell lines 72h after transfection of any of the three siRNAs (Fig 6B), BrdU assays, which were performed 48h post transfection and directly assessed DNA synthesis rates as a measure of cell proliferation, showed a more differentiated picture. PaTu 8988t cells, which have a lower level of endogeneous ADRBK1 expression than S2-028 or HEK293 cells, were most strongly affected by the knockdown, resulting in more than 40% reduction in proliferation activity for all three siRNAs compared to cells transfected with non-silencing control siRNA (Fig 6C, red bars). S2-028 cells showed an intermediate phenotype, with varying but statistically significant reduction of proliferation rates for the individual siRNAS (Fig 6C, blue bars), while HEK293 cells were completely unaffected by the treatment (Fig 6C, green bars). Flow cytometry analyses of PaTu8988t cells with and without ADRBK1 knockdown confirmed the strong impairment of proliferative activity, demonstrating a dramatic reduction of the proportion of cells in S-phase and a strong increase of cells in G1 suggestive of a G1/S cell cycle arrest (Fig 6D).

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Fig 6. Inhibition of endogeneous ADRBK1 expression impairs cell growth.

A: Transfection of three individual siRNAs against ADRBK1 resulted in at least 70% reduction of ADRBK1 mRNA levels in two pancreatic cancer cell lines with high (S2-28) and intermediate (PaTu-8988t) levels of endogeneous ADRBK1 expression, as well as the non-transformed HEK293 cell line. mRNA levels were determined by qRT-PCR and normalized to non-silencing control siRNA (“siControl”). B: MTT assays showed significantly reduced numbers of viable cells 72h after transfection of ADRBK1-specific siRNAs as compared to non-silencing control siRNA. C: PI-staining and flow cytometry analyses were performed 48h after siRNA transfection into PaTu 8988t cells. The results demonstrate strongly increased proportions of cells in G1 phase and strongly decreased proportions of cells in S phase after ADRBK1 knockdown, while G2 phase remained essentially unchanged. Shown is one representative example of three independent experiments.E, F: PARP cleavage was analyzed by Western Blot analyses 72h after transfection of ADRBK1-specific and control siRNAs, respectively. The results showed slightly elevated levels of cleaved PARP protein in S2-028 cells (E, right panel), but this was not apparent in PaRu-8988t (E, left panel) or HEK293 (F) cells. * p<0.05, ** p<0.01 (Student’s t-test).

https://doi.org/10.1371/journal.pone.0122946.g006

In order to assess whether inhibition of ADRBK1 expression also induces apoptosis in the cells, we performed Western Blot analyses in ADRBK1 knockdown and control cells. While ADRBK1 knockdown resulted in slightly elevated levels of PARP-1 cleavage, an indicator of apoptosis, in S2-028 cells (Fig 6E, right panel), levels of cleaved PARP-1 protein were not appreciably elevated above control levels in PaTu 8988t cells (Fig 6E, left panel), and were completely absent in HEK293 cells (Fig 6F). Analysis of caspase-3 cleavage showed similar results (data not shown), indicating that apoptosis induction is not a major downstream effect of ADRBK1 inhibition in PDAC cell lines.