Cytokines secreted by transfected MDA-MB-231 cells have an effect on angiogenesis.

Images taken at 4× magnification of calcein labeled tubes formed by HUVECs transfected with either (a, b) SM20 or WT15 (c, d) aptamer and grown in conditioned media from MDA-MB-231 cells. The number next to each aptamer type indicates the concentration of the aptamer (0 or 100 pM). (e-k) Morphological parameters assessed from images of the tube formation assay. Each plot indicates the difference in the parameter as a function of aptamer type (i.e. SM20 vs. WT15) or aptamer concentration (i.e. 0 vs. 100 pM).</p

Expression of RNA aptamers in MDA-MB-231 cells.

<p>(<b>A</b>) MDA-MB-231 cells were transfected with the aptamers (Sel2, SM20, and WT15). Total RNA was isolated from transfected (+) and non-transfected (-) cells, then RT-PCR analysis were performed. Expression of the aptamer, PAI-1, uPA, uPAR, and β-actin is shown. This experiment was repeated at least three independent times with similar results. (<b>B</b>) The transfected and non-transfected cells were plated in a 96-well dish and allowed to grow in serum containing media for 72 hours post-transfection. The first time point (0) was assessed at 24 hours post transfection. An MTT assay was performed 24 hour intervals. The data was normalized to control cells (set at 100%) at each given time point. Each data point was performed in triplicate. The experiments were repeated at least twice with similar results.</p

Intracellular aptamers inhibit uPA activity.

<p><b>(A)</b> Intracellular uPA or (<b>B</b>) PAI-1 protein levels in cellular extracts of transfected and non-transfected MDA-MB-231 cells were analyzed by Western blot using an antibody to either uPA (A) or PAI-1 (<b>B</b>). (<b>A</b>) Top panel (short exposure) and the lower panel (longer exposure). Total protein concentration was determined and 21 μg protein was added at each experimental condition. The upper band corresponds to the PAI-1/uPA complex (<b>A-B</b>). (<b>C</b>) Intracellular uPA activity was determined in cellular extracts using a chromogenic assay in non-transfected cells (0) and cells transfected with 100 pmol RNA aptamers. Each experiment was performed at least three times with comparable results. **p<0.01, *p<0.05.</p

Expression of RNA aptamers in HUVECs.

<p>(<b>A</b>) Total RNA was isolated from transfected (+) and non-transfected (-) cells, then RT-PCR analysis were performed. Expression of the aptamer, PAI-1, and β-actin is shown. N.B. The SM20 was assay was run separately and then added to the figure. (<b>B</b>) HUVECs transfected with aptamers (Sel2, SM20, and WT15) or non-transfected cells were added to vitronectin coated plates and incubated for 1 hour at 37°C. The non-adherent cells were removed and the adherent cells were assessed by an MTT assay analysis. The percent of adherent cells were normalized to the percent of cells adhering in the absence of aptamers. All reactions were done in triplicates and repeated at least twice times; error bars represent the standard deviation of the data. *p<0.05.</p

Summary of Morphological Data from HUVEC Tube Formation Assay.

<p>Summary of Morphological Data from HUVEC Tube Formation Assay.</p

Levels of secreted cytokines in the conditioned medium of transfected and non-transfected cells.

<p>Conditioned medium from cells transfected with either SM20 or WT15 and non-transfected cells were collected and assayed for cytokines expression as detailed in Materials and Methods. Data represent the average of three to four independent transfection experiments. Error bars represent the standard deviation of the data.</p

Effects of RNA aptamers on migration and invasion of MDA-MB-231 cells.

<p>MDA-MB-231 cells transfected with Sel2 (<b>A</b>), SM20 (<b>B</b>), and WT15 (<b>C</b>) were added to transwell inserts. For migration assays, the cells were added to uncoated transwell inserts and allowed to migrate for 18–24 hours at 37°C. For invasion assays, the cells were added to transwell inserts coated with Matrigel. The cells were allowed to invade for 24 hours at 37°C. Chemo attractants were added to the lower well. Results shown represent the average +S.D. from three independent assays that were performed in duplicate. All data were normalized to migration or invasion in non-transfected cells, which was set at 100%. *<i>p</i><0.05 compared with PAI-1 alone. Each data point was performed in triplicates and the experiments were repeated at least three times with similar results. *p<0.05, **p<0.01.</p

Effects of the RNA aptamers secreted uPA activity and on adhesion of MDA-MB-231 cells to vitronectin.

<p><b>(A)</b> Conditioned medium from MDA-MD-231 cells was collected and assayed for uPA activity as detailed in the Materials and Methods section. <b>(B)</b> MDA-MB-231 cells transfected with aptamers (Sel2, SM20, and WT15) or non-transfected cells were added to vitronectin coated plates and incubated for 1 hour at 37°C. The non-adherent cells were removed and the adherent cells were assessed by an MTT assay analysis. The percent of adherent cells were normalized to the percent of cells adhering in the absence of aptamers. All reactions were done in triplicates and repeated at least three times; error bars represent the standard deviation of the data. No significant difference was observed in any on the treatment groups compared to non-transfected cells.</p

Transfected aptamers in HUVECs decrease tube formation.

<p>HUVECs were transfected with the various aptamers. Forty-eight hours post-transfection, the cells (1.5x10⁴) were placed on matrigel and incubated at 37°C. Tubes formed within 24 hours. The slides were photographed (<b>A</b>) and the total number of tubes was counted by a blinded mechanism (<b>B</b>). Data represent the average number of tubes formed per well from three independent experiments performed in duplicates. Error bars represent the standard deviation of the data. Representative photos are shown. *p<0.05, **p<0.01.</p