Additional file 2 of Thresher: determining the number of clusters while removing outliers

R Code for Analyses. This is a zip file containing all of the R code used to perform simulations and to analyze the breast cancer data. (ZIP 407 kb

Additional file 1 of Thresher: determining the number of clusters while removing outliers

Using the Thresher Package. This file is porovided as a PDF file illustrating the use of the Thresher package with soime simple examples. (PDF 153 kb

DNA damage induced apoptosis is regulated by CLPTM1L.

<p>A) Annexin V binding by flow cytometry of A549 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. B) Relative caspase 3/7 activity in H838 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. Error bars represent one standard deviation from the mean. **- p<0.01 * - p<0.02 by two-tailed Student’s T-Test. C) Micrographs of A549 cells with CLPTM1L knockdown after 24 hours treatment with 50<b> </b>µM cisplatin showing increased genotoxic cell death upon loss of CLPTM1L.</p

Expression of CLPTM1L is increased in lung adenocarcinomas and in lung tumor cell lines.

<p>A) CLPTM1L transcript accumulation as measured by qPCR in lung adenocarcinoma tissues relative to the mean of matched normal tumor adjacent tissue in 30 patients demonstrating a 2.23 fold average increase in expression in tumor tissues. B) CLPTM1L transcript accumulation as measured by microarray in lung tumor cell lines relative to the mean of non-transformed immortalized cell lines demonstrating a 2.02 fold average increase in expression in tumor cell lines. C) Cell line expression data excluding those tumor cell lines with copy number variation demonstrating a 1.83 fold increase in tumor cell lines. D) Cell line divided into adenocarcinoma cell lines and small cell lung cancer cell lines. Black bars represent average values. p-values were obtained using a two-tailed Student’s T-Test.</p

Modulation of Bcl-xL expression is required for apoptotic effects of CLPTM1L.

<p>A) Western blotting for apoptotic regulators in A549 cells with CLPTM1L knockdown after treatment with cisplatin showing decreased Bcl-xL expression upon loss of CLPTM1L. Bcl-xL blots for two separate representative clonal populations of A549 cells with CLPTM1L knockdown (#1 and #2) are shown. B) Graphic representation of Bcl-xL expression in cells with CLPTM1L knockdown from three separate clonal populationsA549. Error bars represent one standard deviation from the mean. * - p<0.003 by Student’s T-Test C) Western blots confirming expression of exogenous Bcl-xL in A549 cells with CLPTM1L knockdown. D) Relative viable cell counts in A549 cells with CLPTM1L knockdown and ectopic Bcl-xL expression after treatment with cisplatin demonstrating the abolition of apoptotic effect of CLPTM1L loss upon ectopic Bcl-xL expression.</p

Principal component analysis demonstrates that LSC, CD34+ and bulk cells are distinct.

<p>Panel. A) Demonstrates separation along principal components 1 (x-axis) and component 2 (y-axis) for the 5 subgroups of samples studied. From the spatial separation it is clear that CD34+ cells (green) occupy a separate space from CD34- cells (blue) from which they were derived, which largely overlap with the unfractionated Bulk population (red). Similarly after the CD38 sort the CD34+CD38+ cells (orange) largely overlay the CD34+ cases, from which these cells were separated, and the CD34+CD38- population (purple) occupies a separate space. B) Principal component analysis based on components 1, 2 and 3 shows clear separation between Bulk (red) CD34+ (green) and CD34+CD38- (purple) samples. </p

Two-way hierarchical clustering of paried differences between A) CD34+CD38- cells and Bulk cells and B) CD34+CD38- cells and CD34+, using normalized data.

<p>Case numbers are listed along the x-axis and protein names along the y-axis. Colors in the heatmap represent log ratios of protein expression in paired samples, with black representing 0, pure red representing +3, and pure green representing -3. Data beyond these bounds was truncated for display purposes. Comparable figures for CD34+ vs. BULK cells, CD34+ vs. CD34- and CD34+CD38+ vs. CD34+CD38- cells are shown as Figures S2C, D and E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078453#pone.0078453.s001" target="_blank">File S1</a>.</p

Summary of t-test results for individual proteins.

<p>The 5 cell subsets are labled as follows along the bottom: Stem = CD34+CD38-, OTHER = CD34+CD38+, POS= CD34+, NEG=CD34+ and BULK= Leuekmia enriched CD3/CD19 depleted cells. These are combined to show the comparison made, e.g. “PosBulk” is a comparison of CD34+ vs Bulk, “StemOther” is a comparison of Stem cells to CD34+CD38+ cells, etc. Proteins are clustered based on the t-statistics from pairwise comparison. Each column has data for the comparison between two subset populations listed at the bottom. Black values represents a t-statistic of zero. Red values are higher in the first of two groups named at the bottom of the column, and green values are higher in the second group. The t-statistic was calculated in each of pairwise comparisons per protein and the range of t-statistics is [-15.4, 22.7]. The values beyond [-15, 15] were chopped for the display purpose only. </p

Hierarchical cluster analysis of paired proteins.

<p>Clustering based on all protein pairs for CD34+CD38- vs. Bulk (p ≤ 10^-10). The same two overall clusters appeared in the paired protein analysis compared with the individual protein comparisons (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078453#pone-0078453-g002" target="_blank">Figure 2A</a>) except for two patients (ID 59 and ID 60). Cluster analysis was also performed on patients grouped by individual protein pathways (e.g., apoptosis, PI3K), and two or three different clusters emerged (See Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078453#pone.0078453.s001" target="_blank">File S1</a>).</p

Comparison of expression in Bulk, CD34+ and LSC to that of normal CD34+ bone marrow cells.

<p>The percentage of BULK (Left), CD34+ (middle) or CD34+CD38- cells (right) samples that were below, within, or above the range defined by normal CD34+ cells is shown. Proteins are ranked so that those with the highest (lowest) expression relative to the normal CD34+are at the top (bottom).</p