Stability analysis.

<p> as a function of for fixed values of For (green circle) the real part of Q takes, depending on a value in the interval The values for x are increasing from A-D, while and remain unchanged. For relatively low values of (A-B) the real part is positive for After one or several turns, i.e by increasing the spiral can potentially cross the origin only once (empty circle). In A the spiral misses the origin, while in B the spiral crosses the origin after one turn. Crossing of the origin means that the corresponding complex number is a root of Q. In C the spiral starts at the origin. This represents the only real positive root of Q. For initially negative values of (D) the spiral can never cross the origin because the distance to the center point (gray circle) is already in the beginning for larger than the distance between the latter and the origin. By increasing y this distance will even grow further according to Eq. 33.</p

DAPI-BrdU pulse-chase labelling FACS data.

<p>Samples taken at several time points after pulse labelling proliferating U87 human glioblastoma cells with The four gated populations are and which are defined precisely in the main text. Briefly, the subscript indicates the phase at the instant of labelling, while the superscripts ‘u’, ‘lu’ and ‘ld’ refers to cells ‘unlabelled’, ‘labelled and undivided’ and ‘labelled and divided’, respectively. The data was generated as described in the Experimental Methods section.</p

Analysis of simulated dual pulse labelling data.

<p><b>A</b>: Average kinetics of unlabelled (dashed line) and labelled cell cohorts (colored lines) were computed from Eq. 25, using ML parameter estimates from the U87 and the V79 data sets (U87: V79: units are hours). Support points and repeats were chosen according to the real experiments. Multinomial noise was added, mimicking the residuals found in the original data sets (see the Computational Methods section for more details). Finally, model solutions (lines) were fitted to the synthetic data sets (triangles). Best fit parameters (U87: V79: units are hours) <b>B</b>: ML parameter estimates from simulated data. All ML regions converge to point estimates (arrows). Squares indicate parameters used for generating the data (see A). <b>C</b>: Bayesian bi-variate 99%-credibility regions for the parameters and for each phase, based on the artificial data.</p

Model based parameter estimation.

<p><b>A</b>: Best fit of the model predictions (lines) to experimentally determined cell fractions after BrdU pulse labelling (dots). U87: In vitro cultured U87 human glioblastoma cancer cell line (three replicates). V79: In vitro cultured V79 Chinese hamster cells (single replicate) (courtesy G. Wilson). Best fit parameter values used to compute model predictions (U87: V79: units are hours). <b>B</b>: Approximate ML regions for the parameters and associated to each phase (gray: red: green: ). <b>C</b>: Bayesian bi-variate 99%-credibility regions for the parameters and for each phase. Arrows indicate point estimates and the dashed lines delineate the information that could have been gained in our <i>thought</i> experiment under noise-free conditions from two support points, one at and a second at . The U87 data set was generated as described in the Experimental Methods section. The V79 data set was a kind gift of G. Wilson.</p

Dual pulse protocol.

<p><b>A</b>: Simplified schematic representations of the protocols corresponding to a conventional single pulse labelling with one nucleoside analog (e.g., BrdU) and a dual pulse labelling experiment with two different nucleoside analogs (e.g., BrdU together IdU or EdU). <b>B</b>: Artificial staining of single-pulse labelling data (for original data see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003616#pcbi-1003616-g002" target="_blank">Fig. 2</a>), showing eight of the nine subpopulations that could potentially be identified with double-pulse labelling. Notice that the four population and that can be followed by the conventional protocol, have each been subdivided according to the cell cycle phases. The naming convention for the populations is as follows: the superscript ( = ‘labelled undivided’,  = ‘labelled divided’,  = ‘unlabelled’) indicates whether the population is labelled and whether it has divided since the time of the first pulse; the first and the second subscript () stand for the phase in which the population was at the time of the first and the second pulse respectively. Double subscripts are used only when necessary.</p

KLF8 and Ki67 expression in non-CNS tumors.

<p>KLF8 (A, C, E) and Ki67 (B, D, F) protein expression was analyzed in some additional tissue samples of breast cancer metastasis (A, B), meningioma (C, D) and non-neoplastic brain (E, F). All tissue samples analyzed confirmed ubiquitous expression of this transcription molecule, irrespective of the assumed proliferation rate.</p

PCR and WB of KLF8 after shRNA knockdown in U87-MG.

<p>(A) U87-MG cells transfected with either scrambled (scr) or KLF8-shRNA (kd) were cultivated for up to 3 days (day 0 = day of seeding). Cells were harvested every 24 hrs and RNA was isolated, transcribed into cDNA and amplified by qPCR; data were normalized relative to levels of the house keeping gene TBP. Semi-quantitative qPCR displayed a clear knock-down in KLF8 expression already 48 hrs after transfection (day 0). Expression levels decreased to about 10% in KLF8-shRNA treated cells compared to cells treated with scrambled shRNA on day 2 after seeding. (B) Subsequent Western Blot analysis of the nuclear fraction of KLF8-kd U87-MG cells on day 4 after seeding revealed that KLF8 protein was still detectable in all transfected U87-MG cells but only to a small extent in the KLF8-knockdown cells indicating that shRNA-knockdown was successful in these transfected cells in concordance with the qPCR results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030429#pone-0030429-g005" target="_blank">Figure 5A</a>).</p

Western blot and densitometric analysis of KLF8 Western blots in gliomas.

<p>Western blot was performed for samples from gliomas of different WHO grades (GBM °IV, LGG °II) as well as non-neoplastic brain (NNB) (A). Densitometric analysis of the Western blot revealed no significant difference in expression of the transcription factor KLF8 in GBM compared to LGG and non-neoplastic brain samples. Protein expression was normalized to the house-keeping gene β-actin (set as 1.0, B).</p

Quantitative PCR.

<p>Samples of low-grade gliomas LGG and GBM (n = 10 each) were analyzed for KLF8-mRNA regulation. Compared to non-neoplastic brain (set as 100%), qPCR did not show any significant difference in the amount of KLF8-mRNA in LGG (97.1%) and GBM (99.3%).</p