DNA condensation by VP19C.

<p>The morphology of the VP19C-DNA complexes was analyzed by electron microscopy. Purified VP19C (150 µg) was mixed with the BamH1 K DNA (250 ng) substrate and the NPC examined after 15 min of incubation. In panel A, full-length VP19C was mixed with DNA. Toroids of different sizes were detected and are indicated by black arrows. In panel B, VP19C N-ter⁷² was incubated with DNA. DNA loop structures (white arrowheads) along with aggregated condensed DNA (marked by black arrows) were induced by VP19C N-ter⁷². Shown in panel C and D is the morphology of DNA in the presence of GST protein or no added protein, respectively. Scale bars are 100 nm and 10 nm for panel A inset.</p

Effect of salt concentration on nucleo-protein complex formation.

<p>Purified VP19C (3 µg) was mixed with the BamH1 K radiolabeled probe (1.5 ng). The formation of a NPC was detected in the first lane when NP-complex forming buffer containing 30 mM NaCl was used for the assay. In similar experiments the NaCl concentration in the NP-complex forming buffer was increased to 0.1, 0.5 and 1.0 M. NPC formation was abolished completely in the presence of 1.0 M NaCl. An arrow on the left side of the panel indicates the VP19C-DNA NPC.</p

DNA binding activity of VP19C resides in the N-terminal 72 amino acids.

<p>A. Purified VP19C N-ter⁷² (6.4 µg) or full-length VP19C (3 µg) was mixed with the BamH1 K radiolabled probe (1.5 ng) and complex formation analyzed by EMSA. Two distinct NPCs were detected when VP19C N-ter⁷² was used. To compare NPC formation with the full-length VP19C, we performed EMSA with full-length protein (lane 2). B. Similar reactions were also performed with wild-type VP19C N-ter⁷² and mutant VP19C N-ter⁷² polypeptides specifying alanine-substitution mutations in the R-rich boxes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104640#pone-0104640-g001" target="_blank">Fig. 1</a>). The arrows on the sides of panels A and B indicate the positions of the NPC in the gel.</p

Amino acid sequence analysis of HSV-1 VP19C.

<p>Amino acid sequence analysis of HSV-1 VP19C.</p

Distribution of the outer diameter of DNA toroids.

<p>Digital electron micrographs of toroids induced by full-length VP19C were examined and a histogram of the distribution of outer diameters of 74 measured complexes were plotted. The mean outer diameter of the toroids was 9.1 nm, with a standard deviation of 2.2 nm.</p

Micro-RNA-125b partially regulates CRMP.

<p><b>A.</b> Western blot analyses showing the expression levels of EMT markers in PDAC cells in which miR-125b expression was determined. <b>B.</b> TaqMan qPCR analysis showing the relative expression of miRNAs (miR-125b and miR-30d) in different PDAC cells. MiR-30d is used as a control. <b>C.</b> Induction of miR-125b expression in BxPC3 upon treatment of gemcitabine. Quantitative PCR (TaqMan) were performed after 72 hrs of incubation with the drug in order to monitor the expression level of miR-125b in BxPC3 cells. Data indicates that expression of miR-125b is increased by the treatment of gemcitabine in a dose dependent manner.</p

A miRNA Signature of Chemoresistant Mesenchymal Phenotype Identifies Novel Molecular Targets Associated with Advanced Pancreatic Cancer

<div><p>In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (<i>BAP1</i>, <i>BBC3</i>, <i>NEU1</i>, <i>BCL2</i>, <i>STARD13</i>). Thus far, two of these target genes, <i>BBC3</i> and <i>NEU1</i>, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.</p></div

Primers and oligonucleotide sequences used in this study.

a<p>yeast homology sequences are italicized.</p>b<p>restriction sites are underlined and italicized.</p>c<p>alanine substitutions are underlined.</p

VP19C binds non-specifically to dsDNA probes.

<p>Three different nucleic acids were used in the EMSA; HSV-1 DNA (BamH1 K, 5.9 kb; lanes 1–6), plasmid pUC19 (2.7 kb; lanes 11–14) that was linearized by EcoR1 and an oligonucleotide (28mer; lanes 7–10) to test the specificity of the binding. All DNA molecules were 5′-end-labeled with [γ-³²P] ATP. In lanes 1, 7 and 11, dsDNA or oligonucleotide probe alone was added to the assay (1.5 ng). In lanes 2-4 the DNA probe was incubated with increasing concentrations of VP19C (1.6 µg, 3.2 µg and 6.4 µg respectively) in NP-complex forming buffer. Similarly in lanes 8 and 12, 1.6 µg of VP19C was used and in lanes 9 and 13, 6.4 µg of VP19C was used. Lane 6 conditions were the same as lane 4 but the NP-complex forming buffer was free of ZnCl₂. In lanes 5, 10 and 14 the DNA-probe was incubated with 6.4 µg of purified GST.</p

Purification of VP19C and the DNA-binding property of the purified protein.

<p>A. Expression and purification of VP19C protein from yeast cells. Yeast cells induced for expression of GST-VP19C and the proteins purified from these cells were analyzed by SDS/PAGE using 4-12% Nu-PAGE gels and detected by Coomassie Blue staining. Total proteins extracted from uninduced cultures (Un) and cells induced (In) with glucose are shown. The protein fractions, flow-through (FT) and wash (Wash) obtained during the purification are also shown. GST-VP19C bound with glutathione–Sepharose beads before elution (Beads) and the eluted purified protein (Elution) were detected. Molecular-mass markers; lane 2. B. VP19C specifies a DNA-binding property. In the EMSA a small dsDNA (66 bp) radiolabeled probe (1.5 ng) was incubated with 1.5 µg (lane 2) and 3 µg VP19C (lanes 3 and 4) or no protein (lane 1). In lane 4, a 50-fold excess of non-labeled DNA was added to the VP19C-DNA composition. The nucleo-protein complex (NPC) and the location of the free probe are indicated on the left of the panel.</p