Flow cytometry binding assay of selected FITC labeled ssDNA pools with mTECs.

<p>Enrichment in binding random ssDNAs in the selection was observed by flow cytometry analysis. Here the curve represents non-treated cells and treated cells with a zero cycle ssDNA 10 and 12 cycle ssDNA pools respectively. 400 pmol of ssDNA library was used in each case. A zero-cycle ssDNA pool was used to observe the differentiation of enrichment from the starting point of selection with increasing cycle of selection.</p

<i>In vitro</i> cellular uptake of the AraHH001 aptamer and control zero cycle pools in acidic compartments of mTECs.

<p>mTECs was incubated with FITC-tagged aptamer AraHH001 and zero cycle pools at 37°C for 30 minutes to check internalization, and were stained live nuclei with Hoechst 33342 and acidic compartments with LysoTracker Red and analyses with confocal laser scanning microscopy. <b>A.</b> (FITC-tagged aptamer AraHH001 and zero cycle pools). <b>B. (</b>Uptake of Lysotracker red into the acidic compartments of mTECs). <b>C.</b> (merge of images, Hoechst, A, and B). <b>D.</b> (merge of image Hoechst, A and B+DIC).</p

Binding assay of selected FITC-labeled DNA aptamer AraHH001 against a series of cells, and cell lines by a flow cytometry assay.

<p>A flow cytometry binding assay of AraHH001 with A. Normal skin-ECs, B. OS-RC-2, C. RFP-SM, D. HUVEC, E. HMVEC, F. m OS-RC-EC, and G. hTEC. In all cases, the results show non-treated, treated with 200 pmol FITC labeled zero cycle ssDNA pools, treated with 200 pmol and 1000 pmol FITC-labeled DNA aptamer AraHH001.</p

Identification of selected FITC labeled DNA aptamers on mTECs by a flow cytometry assay.

<p>Flow cytometry data represents binding assay of five FITC-labeled DNA aptamers with mTECs. In each DNA aptamer binding assay has shown the result of non treated mTECs, treatment mTECs with 200 pmol FITC labeled zero cycle ssDNA pools, treatment mTECs with 200 pmol DNA aptamer and 1000 pmol DNA aptamer independently.</p

Determination of binding affinity, Kd value of the DNA aptamer AraHH001 by a flow cytometry.

<p>The Binding affinity of AraHH001 was determined by a flow cytometry using FITC-AraHH001 to mTECs and to skin-ECs. The average mean fluorescence intensity of varying concentration of FITC-AraHH001obtained was plotted to determine dissociation constant Kd. The experiment was repeated three times and a Error bar represents the standard deviation of means.</p

<i>In vitro</i> tube formation assays to measure anti-angiogenesis activity of the AraHH001.

<p>The tube formation was observed after 20 hours incubation with non-treated, zero-cycle library treated as a negative control, and AraHH001 treated mTECs on matrigel. <b>A.</b> Microscopic observation of tube formation of mTECs on matrigel. The tube formation was prohibited by AraHH001 treated mTECs on matrigel. Scale bar 100 µm. <b>B.</b> Quantitative analysis of tube formation. The tube length was calculated in pixels using one-way ANOVA after SNK test in each sample, and performed statistical analysis<b>.</b> Data are represented as mean± SD (n = 3 in each cases). **P<0.01 show significant differences NT and zero-cycle vs. AraHH001 treated group.</p

A schematic representation of the cell-based SELEX method used for the selection of DNA aptamer.

<p>In short, a 200 pmol ssDNAs library was incubated with mTECs on ice for 45 minutes. A five molar excess BSA and yeast tRNA was used to reduce nonspecific binding. After washing bound ssDNA from cells were eluted by heating at 95°C for 5 minutes. Selected ssDNA pools were subjected to amplify with fluorescent tag to start the next cycle. At cycles 11 and 12, negative selection was done using skin-ECs and OS-RC-2 cell lines along with the positive selection. After a successful 12 cycle’s selection, the enriched pool of ssDNA was subjected to clone and sequence for the identification of the individual aptamer.</p

Karyotypes of HMVECs exposed to hypoxia.

<p>Karyotypes of HMVECs after exposure to normoxia, hypoxia (1% O2), and hypoxia-reoxygenation were analyzed by Q band analysis. A karyotype of one cell in each condition is shown. (A) HMVECs in normoxia were essentially diploid. (B, C) HMVECs exposed to hypoxia and hypoxia-reoxygenation had complex abnormal karyotypes and were aneuploid. (D) The chromosome number of each condition was counted and shown.</p

Assessment of tumor tissue hypoxia and analysis of ploidy in TECs by FISH Hypoxic areas in supermetastatic tumor cryosections were detected using hypoxyprobe.

<p>To analyze the hypoxic area in TECs in in-vivo tumors, frozen sections of tumors were immunostained with anti-CD31 (red) and anti-pimonidazole (green) followed by FISH using a spectrum red-conjugated mouse chromosome-17 locus-specific probe (red spot). Representative CD31-positive ECs in tumor tissue. (A, D, G) The blood vessels in tumor tissue were pimonidazole-positive. (B, E, H) Nuclei are stained with DAPI (blue). Aneuploidy was also observed in ECs of tumor blood vessels exposed to hypoxia. (C, F, I) Pimonidazole-nagative ECs in tumor tissue did not exhibit aneuploidy. (J, K, L) ECs in normal tissue did not exhibit aneuploidy. (M, N, O) Scale bar, 20 μm.</p

Hypoxia induces aneuploidy in HMVECs.

<p>(A, B) After HMVECs were cultured in each condition for 7 days, FISH analysis using a chromosome-7 probe revealed that approximately 22.8% ECs in the hypoxic condition (1% O2) and 19.8% ECs in the hypoxia-reoxygenation condition were aneuploid, whereas 6.4% ECs in the normoxic condition were aneuploid. (C, D) FISH analysis using a chromosome-8 probe revealed that approximately 19.1% ECs in the hypoxic condition and 23.3% of ECs in the hypoxia-reoxygenation condition were aneuploid, whereas 8.5% ECs in the normoxic condition were aneuploid.</p