1. Volume 21, Issue 5, Pages 1004-1013 (May 2013)
Periostin-binding DNA Aptamer Inhibits Breast Cancer Growth and Metastasis 
Yu Jin Lee, Il Shin Kim, Soo-Ah Park, Youndong Kim, Jeung Eun Lee, Dong-Young Noh, Kyong-Tai Kim, Sung Ho Ryu, Pann-Ghill Suh 
Molecular Therapy 
Volume 21, Issue 5, Pages (May 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Characterization of periostin-PNDA interactions. (a) Binding affinities were determined using 32P-labeled PNDAs and varying hPN concentrations. (b) Competition of PNDA binding to hPN. Proteins were incubated with 32P-labeled PNDA-3 and an unlabeled control aptamer or PNDA-3 for 30 minutes, and the protein:aptamer complex was precipitated and quantified. (c) Binding of PNDA-3 to hPN, mPN, hβIG-H3, and hIgG was assayed in triplicate using direct binding assays. A control that contained no protein was also processed via the same method, and the resultant value was set as the background. Binding assays were performed in triplicate, and the means ± SD are shown. (d) Left, untreated 10% serum buffer (lane 1), biotinylated CTL aptamer treatment (lane 2) or PNDA-3 treatment (lane 3) were purified on streptavidin beads and then stained with SYPRO Ruby and immunoblotted. Lane 4, one-tenth of the hPN was loaded as input (I). Right, dose-dependent binding of PNDA-3 to periostin. hPN, human periostin; mPN, mouse periostin; WB, western blot.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
PNDA-3 specifically interacts with periostin. (a) A Cy3-labeled control (pink, 100 nmol/l; green, 500 nmol/l) or PNDA-3 (blue, 100 nmol/l; orange, 500 nmol/l) was incubated with periostin-overexpressing (4T1, left) and periostin-negative cells (MCF7, right) and analyzed by flow cytometry. A control that contained no DNA was also included (black). Counts represent the number of cells counted. (b) Left, binding of 500 nmol/l Cy3-labeled PNDA-3 to 4T1 cells following a 72-hours transfection with a specific periostin siRNA (PN siRNA #1, green) or a nonrelated siRNA (control siRNA, pink). Right, lysates from 4T1 cells following a 72-hour transfection with PN siRNA or a control siRNA were quantified by quantitative real-time PCR and western blotting with an anti-periostin antibody. The blots shown are representative of at least three independent experiments, and anti-β-actin antibodies were used as an internal control. *P < 0.01; **P < 0.05 compared with control siRNA. (c) The cell surface binding of PNDA-3 to 4T1 cells was analyzed by TIRF microscopy. A specific competitor (unlabeled PNDA-3) or nonspecific competitor (unlabeled control aptamer) was incubated with 100 nmol/l Cy3-labeled-PNDA-3 for 15 minutes. Images were acquired after washing. All images were captured using the same exposure. Representative images from five random fields of at least three independent experiments are shown. Scale bar: 20 µm. PN, periostin; TIRF, total internal reflection fluorescence.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
PNDA-3 inhibits periostin-dependent signaling. (a) Schematic representation of the full-length periostin and deletion constructs used in this study. (b) Biotinylated PNDA-3 was mixed with cell lysates from 293T cells overexpressing mutant periostins, and a pull-down assay was performed with streptavidin beads, followed by western blotting with the indicated antibodies. (c) 4T1 and (d) MDA-MB-231 cells were serum starved, untreated, or treated for 3 hours with PNDA-3, a control aptamer or anti-integrin Abs and then stimulated for 90 minutes with periostin (100 ng/ml) in the presence or absence of each aptamer or Ab. Cell lysates were prepared, and the activated forms of phospho-FAK (Tyr397) and phospho-Src were analyzed by western blotting. The total fraction of FAK and Src was used as the experimental control, and β-actin was used as a loading control. The blots shown are representative of three experiments. WB, western blot.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Inhibitory effects of PNDA-3 on breast cancer cell adhesion, migration, and invasion. (a) In the adhesion assay, breast cancer cells were preincubated with PNDA-3, the control aptamer (100 nmol/l) or anti-integrin Abs (5 µg/ml) and then added to periostin-coated 96-well plates. Adherent cells were quantified in a microplate reader at 570 nm. BSA-treated cells were used as a negative control (set as 100%). (b) The effect of PNDA-3 on the migration of breast cancer cells was analyzed using a transwell migration assay in the presence of PNDA, a control aptamer or anti-integrin Abs for 3 hours toward periostin, which was used as a migration inducer. (c) Cell invasion through Matrigel toward periostin was performed in the presence of PNDA-3, a control aptamer or anti-integrin Abs for 24 hours. (b,c) The cells that migrated or invaded were stained with Hoechst and counted with an inverted microscope (10× objective). The values represent the means ± SD of five independent experiments. *P < 0.01; **P < 0.05 compared with the BSA control. BSA, bovine serum albumin; PN, periostin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
PNDA-3 inhibits breast cancer growth and metastasis in vivo. (a) PNDA-3 inhibited tumor growth in a syngeneic breast cancer model employing periostin-positive 4T1 cells (58% at day 16 compared with CTL or the control group); *P < Day 0 marks the first day of injection. The data are expressed as the means ± SD (n = 10 tumors). (b) Mean body weights of the animals. (c) Necropsy was performed on all of the mice, and representative images of treated and untreated mice are shown. White arrows indicate lung nodules (left). The bar graphs demonstrate the number of lung nodules. (n = 10; *P < 0.005) (right). (d) Representative lung tissue sections stained with hematoxylin and eosin demonstrate metastatic foci in the vehicle-, control aptamer-, and PNDA-3–treated groups. The black arrows and circles denote metastatic foci in the lung. Scale bars: 200 µm (top) and 100 µm (bottom).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
In vivo effects of PNDA-3 on tumor growth and angiogenesis. (a) Representative images of primary tumor sections from the vehicle-, control aptamer-, and PNDA-3–treated groups, which were stained as indicated. (b,c) Quantification of the Ki-67 (b) and CD31 (c) staining of proliferating and endothelial cells, respectively. Percent values represent the means ± SD of 10 randomly selected fields. *P < The original images were digitally captured. Scale bar: 100 µm. H&E, hematoxylin and eosin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions