Inhibition of migration of MDA-MB-231 and HCC1806.

<p>MDA-MB-231 (A) or HCC1806 (B) cells were harvested, washed and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The aptamers were added in both upper and lower chambers at the indicated concentrations. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test.</p

CD44 associated with EphA2 on breast cancer cells.

<p>(A) HCC38 cells were lysed with 100 mM Tris-HCl (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, and a protease inhibitor cocktail by scraping and pipetting. The cell lysates were precleared and immunoprecipitated with anti-CD44 antibody clone (clone 156-3C11)-, anti-EphA2- or control IgG-protein G beads for 4 hours at 4°C. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 antibody followed by HRP-secondary antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. (B) Cells were harvested using 5 mM EDTA in PBS and were lysed in 100 mM Tris-HCL (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, and a protease inhibitor cocktail. The lysates were precleared with anti-FLAG (M2) agarose beads for 4 hours by shaking at 4°C. The cleared lysates were incubated with recombinant protein CD44v10 P-FLAG-anti-FLAG (M2) antibody-agarose beads (CD44v10) or anti-FLAG (M2) antibody-agarose beads (Con.) in the presence or absence of aptamers (Apt#4 and Apt#7) by shaking at 4°C for 4 hours. The beads were washed extensively with the lysis buffer. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 followed by HRP-secondary antibody or HRP-conjugated anti-FLAG (M2) antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. CD44 exon v10 peptide was used as a loading control for ensuring the same amount of proteins was loaded in each lane.</p

FACS analysis of aptamers against CD44 exon v10.

<p>HCC38 or SK-Br3 cells were harvested and resuspended in RPMI1640 containing 1 mg/ml BSA and 0.025% NaN₃ (FACS buffer). Cells were incubated with aptamers of which 5′-end was conjugated to biotin (5 µg/ml), anti-panCD44 (clone 156-3C11) (5 µg/ml), or anti-CD44 exon v10 antibody (AB2082) (5 µg/ml) for 1 hour at 4°C. After washing with the same buffer, cells were incubated with Streptavidin-FITC, FITC-goat anti-mouse IgG, or FITC-goat anti-rabbit IgG for 1 hour at 4°C. After extensive washing with FACS buffer, cells were resuspended in PBS containing 1% paraformaldehyde. Expression was measured on FACS Calibur (Becton-Dickinson, NJ USA) by collecting 10,000 events and analyzed by CellQuest.</p

Inhibition of cell migration by aptamers against CD44 exon v10.

<p>(A) HCC38 cells were harvested, washed and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The aptamers were added in both upper and lower chambers at the indicated concentrations in the text. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test. (B) MCF-7 cells were stably transfected with CD44E according to the methods described in Materials and Methods. MCF-7 cells transfected control vector, [MCF-7(pIRES2)], MCF-7 (CD44E), and parental MCF-7 cells (NT) were lysed and subjected to western blotting for the expression of CD44E using anti-CD44 exon v10 antibody followed by HRP-conjugated goat-anti-rabbit IgG. The signal was detected by ECL reactions. Actin was used as a loading control. (C) The migration of these cells was evaluated as described above in the presence or absence of aptamers against CD44 exon v10 at a concentration of 10 nM as described above. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test.</p

Protocol for isolating CD44 exon v10-specific DNA aptamers by Systematic Evolution of Ligands by Exponential Enrichment (SELEX).

<p>The CD44 exon v10 peptide was expressed as a fusion protein with FLAG tag. The purified recombinant peptide was immobilized on plates coated with anti-FLAG (M2) antibody. Library of DNA aptamers were incubated on the plates and extensively washed to remove non-bound aptamers. The isolated aptamers were then amplified and this selection process was repeated 10 times. Aptamers isolated from the last selection were amplified and ligated to pCR2.1-TOPOTA vector for sequencing.</p

Inhibition of migration of HCC38 cells with anti-CD44 exon v10 antibody.

<p>(A) Cells were harvested, washed, and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The antibodies were added in both upper and lower chambers at a concentration of 5 µg/ml. Experiments were performed by triplicates three times. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test. (B) Cells were harvested, washed, resuspended in RPMI1640 containing 1 mg/ml BSA (adhesion buffer) at a concentration of 10⁵ cell/ml. Cells (100 µl/well) were incubated on plates coated with type I collagen (5 µg/ml) for 1 hour in the presence or absence of antibodies (5 µg/ml). Plates were washed, fixed followed by staining with crystal violet. After extensive washing to remove excess dye, cells were lysed with 100 µl of PBS containing 2% SDS and then the absorbance was measured at 550 nm. Experiments were performed in quadruplicates and repeated three times. The representative data were shown as mean +/− S.D. of absorbance values.</p