Data files for figures 1-8

2013-11-11T12:18:36Z (GMT) by Ron Prywes
<p>Data file 1. Data for figures 1A, B and D and data not shown.<br>A, B) Relative mRNA expression from the indicated cell lines is shown for three replicates from independent mRNA isolations. The numbers reflect qPCR delta Ct numbers using 18S rRNA Ct values. The values were scaled to an average of 1.0 for Scp-2 cells. The primers used are shown in Materials and Methods.<br>D) Relative MMP-1 protein expression. Three immunoblots were performed with anti-MMP1 sera and lysates of the indicated cell lines as in figure 1C. The MMP-1 band intensities were quantified using the Odyssey infrared imager (Li-Cor) and the software provided by the manufacturer. The intensities were scaled to the average intensities of MMP-1 in Scp-2 cells.<br>Data not shown) Relative MMP-1 expression in other MDA-MB-231 single cell population cell lines as in figure 1A. Values are from two independent replicates.</p> <p>Data file 2. Data for figures 2C and E.<br>Relative luciferase activity is shown for three replicates. Each replicate is the average of duplicate determinations (i.e. six total points). The firefly luciferase values were divided by the Renilla luciferase values (from cotransfected plasmid pRLSV40P). In (C) the values were scaled to an average of 1.0 for the Scp-2 cells and the -819 to +71 MMP1 reporter, except for CMV which was scaled to its own values for Scp-2 cells. In (E) the values were scaled in each replicate to 1.0 for Scp-2 cells and the -172/-27 MMP1 reporter.</p> <p>Data file 3. Data for figures 3B and D.<br>Relative luciferase activity is shown for three replicates. Each replicate is the average of duplicate determinations (i.e. six total points). The firefly luciferase values were divided by the Renilla luciferase values (from cotransfected plasmid pRLSV40P). The values were scaled in each replicate to 1.0 for Scp-2 cells and the wild type reporter. For the synthetic reporter, the values were scaled to that of the 3X-AP1 site reporter.</p> <p>Data file 4. Data for figures 4A and C.<br>A) Relative mRNA expression from the indicated cell lines and genes is shown for three replicates from independent mRNA isolations. The numbers reflect qPCR Ct numbers using 18S rRNA Ct values. The values were scaled to a value of 1.0 for Scp-2 cells. The primers used are shown in Materials and Methods.<br>C) Relative Fra-1 protein expression. Three immunoblots were performed with anti-Fra-1 sera and lysates of the indicated cell lines as in figure 4B. The Fra-1 band intensities were quantified using the Odyssey infrared imager (Li-Cor) and the software provided by the manufacturer. The intensities were scaled to the average values for Fra-1 protein in Scp-2 cells.</p> <p>Data file 5. Data for figures 5A, C and D.<br>A) Relative Fra-1 mRNA expression in Scp-2 cells treated with the indicated siRNAs. Expression determined by qPCR is shown for three replicates from independent mRNA isolations. The numbers reflect qPCR delta Ct numbers using 18S rRNA Ct values. The values were scaled to an average value of 1.0 for untreated Scp-2 cells. The primers used are shown in Materials and Methods.<br>B) Relative Fra-1 protein expression. Three immunoblots were performed with anti-Fra-1 sera and lysates of Scp-2 cells treated with the indicated siRNAs as in figure 5B. The Fra-1 band intensities were quantified using the Odyssey infrared imager (Li-Cor) and the software provided by the manufacturer. The values were scaled to the average intensities of Fra-1 protein in Scp-2 cells.<br>C) As in (A) exept that MMP-1 and GAPDH mRNA expression were measured by qPCR. The primers used are shown in materials and methods.</p> <p>Data file 6. Data for figure 6C.<br>Chromatin immunoprecipitation from the indicated cell lines and using anti-Fra1 or control (no antibody) was quantified by qPCR using the indicated primers. The binding values were divided by those of input DNA and are shown as percent of input. Values from three replicate chromatin immunoprecipitations are shown.</p> <p>Data file 7. Data for figures 7B and D.<br>B) Scp-2 or Scp-21 cells were treated with the protein synthesis inhibitor cycloheximide for the indictate times. The cell lysates were then immunoblotted with anti-Fra-1 antibodies as in figure 7A. The Fra-1 protein band intensities were quantified on the Odyssey infrared imager (Li-Cor). The values for three independent experiments are shown. The values were scaled to 1.0 for cells treated for 0 hours (i.e. untreated).<br>D) Scp-2 and Scp-21 cells were metabolically labeled with 35S-methionine and –cysteine for the indicated times. Fra-1 protein was then immunoprecipitated with anti-Fra-1 antibodies, run on SDS-PAGE and exposed to film. The bands were quantified using ImageJ software. The values were scaled to the value of Fra-1 expression in Scp-21 cells at 15 minutes of labeling. Values for there independent replicates are shown.</p> <p>Data file 8. Data for figures 8A, D and E.<br>A) Relative MMP-1 mRNA expression in the indicated cell lines with no expression vector, control pBabepuro vector or Fra-1 expressing pBabFra-1 vector. Expression determined by qPCR is shown for three replicates from independent mRNA isolations. The numbers reflect qPCR delta Ct numbers using 18S rRNA Ct values. The values were scaled to an average value of 1.0 for untreated Scp-2 cells. The primers used are shown in Materials and Methods.<br>D) Quantfication of cell motility as in Fig. 8C. The number of cells crossing the initial scratch after 18 hours were counted in three independent experiments.<br>E) Quantification of anchorage independent growth in soft agar. The number of colonies growing after 21 days of plating in soft agar were counted in three independent experiments.</p> <p> </p>