Partial Tpx2 ablation in 6DT1 cells does not change expression levels of Tpx2 network hub genes.

<p>6DT1 mouse mammary carcinoma cells were lentivirally transduced with pLKO-Scrambled (shCtrl), pLKO-Tpx2#1 (shTpx2#1), or pLKO-Tpx2#2 (shTpx2#2) and stable, polyclonal pools generated. qRT PCR was carried out to measure mRNA levels of <i>Tpx2</i> and eight genes previously identified as ‘hubs’ of the Tpx2 gene expression network <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111813#pone.0111813-Hu1" target="_blank">[3]</a>. Gene expression levels are displayed relative to levels in shCtrl control cells, error bars represent standard deviations, asterisks indicate p-values <0.05. Only <i>Tpx2</i> was significantly down regulated in both shTpx2 cell lines (red asterisks).</p

Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.

<p>A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.</p

Network modules from the cross species analysis.

<p>A) Proliferation associated network centered around the <i>Tpx2</i> gene. B) Immune cell network containing the <i>Il10ra</i> gene. Both Figures are adapted from Hu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111813#pone.0111813-Hu1" target="_blank">[3]</a>.</p

Knockdown of Tpx2 does not impair 6DT1 cell proliferation in vivo and does not affect anoikis.

<p>A) Lung sections from mice injected with 6DT1-shCtrl or 6DT1-shTpx2#1 harboring metastatic nodules and immuno-labeled with Ki-67 antibody. The bar diagram on the right represents quantification of percent tumor cell nuclei with immuno labeling (cycling fraction) relative to total numbers of tumor cells from five mice each. Error bars show standard deviations. B) Sections of primary tumors from mice injected with 6DT1-shRNA control or 6DT1-shTpx2#1 were stained and analyzed as in a). Scale bars correspond to 100 µm. C) shCtrl or <i>Tpx2</i> knockdown cells were plated into low adhesion plates, grown for 7 days, and viable cells quantified. The combined results of four independent experiments are represented. No significant difference in anoikis was observed by reduced <i>Tpx2</i> levels.</p

Tpx2 knockdown significantly affects metastasis but not tumor proliferation.

<p>A) Relative expression levels of <i>Tpx2</i> in 6DT1 cells as measured by qRT-PCR and western blotting. B) 1×10⁵ 6DT1-shTpx2 or -shCtrl cells were orthotopically injected into the mammary fat pad of female FVB mice. Mice were euthanized and lungs dissected and inspected for metastatic nodules on the surface of the lungs 27 days after injection. Asterisks indicate a p-value <0.05. C) Weight of primary tumors dissected from the mammary fat pad of mice described in A. No significant differences (N.S.) were observed. D) The Gene expression-based Outcome for Breast cancer Online (GOBO) database was queried for Tpx2 and distant metastasis-free survival (DMFS) plotted as Kaplan-Meier curves for patients with ER-positive tumors expressing high (blue), intermediate (red), or low (gray) levels of <i>TPX2</i>.</p

Knockdown of Tpx2 does not impair 6DT1 cell proliferation in vitro.

<p>A) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells were seeded in triplicate at equal densities and passaged every 3–4 days for 17 days. Cumulative cell numbers were determined at each passage. B) 2000 cells were seeded in quadruplicates into 48-well cell culture plates and imaged for >80h. Average cell density during logarithmic growth from 20 h–80 h after seeding is displayed and exponential trend lines are shown in black. C) Exponentially growing cells were pulsed with 10 µM BrdU for 25 min, stained for DNA and BrdU content and analyzed by FACS. Percentage of cells in G1, S, and G2/M phase are indicated.</p

Reduction of Tpx2 does not increase wound-healing and apoptosis in 6DT1 cells.

<p>A) Representative photomicrographs of the shCtrl, <i>Tpx2</i> sh1 or <i>Tpx2</i> sh2 cells in the wound healing assay are shown at the top of the figure. A graphical representation of the assay, portrayed as percent confluence over time, is shown at the bottom of the figure. B) Percent of cells with positive labeling in the lung metastases for TPX2 control and knockdown lungs. For each sample, 4 quantification areas were set up in different metastatic nodules. Two knockdown samples did not have 4 metastatic nodules. In one sample, only one nodule is present. In the other sample, there were 3 nodules counted. Counting areas included either 2000–4000 cells or the entire nodule if it was less than 2000 cells. Areas with apparent off-target labeling due to necrosis were avoided, but single cell necrosis may be contributing to the increased labeling in the control metastases.</p