Additional file 1: of Real-Time Vital Mineralization Detection and Quantification during In Vitro Osteoblast Differentiation

Figure S1. a An example of the mask set for the green objects count on day 10; left pictures shows green fluorescent dots corresponding to the forming calcium crystals; right pictures shows how the IncuZOOM mask depicted green dots with high sensitivity (even very weak and small green dots are counted), representative pictures are shown. b Real-time calcium crystals formation detected by fluorescent imaging in calcein-treated and calcein-untreated samples, n = 3, SE. c Calcein detection in negative control and PPP2R2C siRNA treated samples, representative pictures are shown. d Number of dead cells detected by propidium iodide in calcein-treated and calcein untreated cells, n = 2, SE. (TIF 2900 kb

Endogenous transcript levels of mature miR-142-5p/3p and primary <i>mir-142</i> in cells of mesenchymal and hematopoietic origin, and after treatment with epigenetic modifiers.

<p>(<b>A</b>) Expression levels as determined by qPCR in tumorigenic cells (MG-63, OHS, IOR/OS14, U-2 OS, IOR/OS10, IOR/SARG, IOR/MOS and HOS), non-tumorigenic mesenchymal cells (iMSC#3, hMSCs and primary osteoblasts), and hematopoietic cells (K562 and PBPCs). Expression is depicted relative to the MG-63 cells (set to 1) and the numbers above the histograms represent the expression level of primary <i>mir-142</i>. <i>RNU44</i> or Glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH</i>) were used for normalization of mature and primary transcripts, respectively. iMSC#3, immortalized bone marrow-derived stromal cells; hMSCs, primary bone marrow-derived stromal cells; K562, K562 leukemia cells; PBPCs, peripheral blood progenitor cells. (<b>B</b>) Quantification of expression using qPCR after treatment with 5-Aza-2′-deoxycytidine (5-Aza). Expression is shown relative to untreated cells (set to 1). <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts, respectively. (<b>C</b>) Expression levels of miR-142-3p, primary <i>mir-142</i> and mesoderm specific transcript (<i>MEST</i>) in MG-63 cells as determined by qPCR, after treatment with 5-Aza alone, a combination of 5-Aza and trichostatin A (TSA), or TSA alone. <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts (including <i>MEST</i>), respectively. The error bars in all qPCR experiments show the standard deviation of technical replicates.</p

Analyses of methylation status of CGIs associated <i>mir-142</i> and <i>in vitro</i> methylation of its upstream regulatory region.

<p>(<b>A</b>) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. (<b>B</b>) Schematic representation of the <i>mir-142</i> locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#pone-0079231-g003" target="_blank">Figure 3B</a> for more information). E-box, enhancer box. Numbers indicate the position relative to <i>pre-miR-142</i> (+1 to +87). (<b>C</b>) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#s2" target="_blank">Materials and Methods</a>. A <i>P</i> value ≤0.05 was considered as significant. (<b>D</b>) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. (<b>E</b>) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). (<b>F</b>) The 2,031 bp upstream region of <i>pre-mir-142</i> was cloned into the promoter-less luciferase reporter construct pCpGL-basic and <i>in vitro</i> methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a <i>Renilla</i> reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the <i>P</i> values are shown above the histograms. A <i>P</i> value ≤0.05 was considered as significant.</p

<i>In silico</i> identification of putative CGIs and characterization of the primary <i>mir-142</i> transcript.

<p>(<b>A</b>) Schematic representation of putative CGIs associated with <i>mir-142</i>. Each vertical line represents an individual CpG. Black, dark grey and light grey horizontal bars; CGIs predicted with CpG Island Explorer, CpG Island Searcher and CpGcluster, respectively. The numbers indicate the position relative to the <i>mir-142</i> precursor (<i>pre-mir-142</i>) (+1 to +87). O/E, observed/expected. (<b>B</b>) Schematic representation of the genomic <i>mir-142</i> region. The approximate 5′- end of <i>mir-142</i> was mapped using qPCR with assays specific for four different regions (depicted by vertical arrowheads). Grey and black arrowheads indicate increased expression or no change, respectively. The transcription start (TSS) and polyadenylation sites were identified using rapid amplification of cDNA ends. The approximate positions of the gene-specific primers (GSPs) and associated amplicons are indicated by horizontal arrowheads and attached lines, respectively. The putative CGIs are represented by horizontal bars. (<b>C</b>) Schematic representation of the 2 transcript variants of primary <i>mir-142</i> (<i>pri-mir-142</i>).</p

Generation and Characterisation of Novel Pancreatic Adenocarcinoma Xenograft Models and Corresponding Primary Cell Lines

<div><p>Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection <i>in vitro</i>, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding <i>in vitro</i> cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease.</p></div

Colony forming capacity of the cell lines.

<p>Each diamond represent the fraction of cells able to generate colonies >50 µm in methylcellulose/stem cell medium II in one experiment (n = 3–9). The red line shows the average and the yellow line shows the median colony forming capacity for cells in the following passage span: PpaC1 p1–p31, PpaC2 p5–p13, PpaC6 p2–p7 and PpaC8 p4–p14.</p

Characterisation of patient material.

1<p>: PDAC = Pancreatic ductal adenocarcinoma, P = pancreatobiliary subtype, I = intestinal subtype.</p><p>IPMN = Intraductal papillary mucinous neoplasia.</p>2<p>: The degree of differentiation for each tumour is according to the pTNM Classification of Malignant Tumours <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103873#pone.0103873-Sobin1" target="_blank">[14]</a>.</p>3<p>: T3 = Tumour extends beyond pancreas, but without involvement of celiac axis or superior mesenteric artery, Tis = Carcinoma <i>in situ</i>.</p>4<p>: 0 = resection margin free and 1 = resection margin not free.</p>#<p>: Patient only followed up the first year after operation, due to non-malignant disease.</p><p>*: Patient is alive, the number of days given refers to the latest follow-up date.</p

Global mRNA expression pattern.

<p>Heat map showing the hierarchical clustering of normal pancreatic tissue, original tumour material and the corresponding cell lines. The data set consists of 10 948 genes after filtering genes commonly regulated in fresh samples or cell lines. The RNA is isolated from the cell lines at the following passages: PpaC1 p16, PpaC6 p11 and PpaC8 p16.</p

Colony forming capacity of PpaC1.

<p>The table shows the % of cells able to generate colonies in the indicated semi-solid media in different <i>in vitro</i> passages, n.d. = not done.</p

Cell surface expression of known cancer and stem cell markers.

<p>Shown here is the average % positive cells out of single, live cells, n = at least 3, n.d = not done.</p><p>Cells were analysed in the following passage spans: PpaC1 p1–p10, PpaC2 p3–p14, PpaC6 p5–p9 and PpaC8 p4–p20.</p