Detection of α-syn pores.

<p><b>A</b>) Pore formation results in increased current-flows over the membrane (black trace) compared to intact bilayers (grey trace) when a voltage ramp is applied. <b>B</b>) Pore detection rate for oligomers obtained with 2.1 µM α-syn with 1% DMSO and 20 µM Fe³⁺ at RT. 4 and 24 h of incubation did not lead to any pore detections (N = 8, each). In 33% of all 48 h-samples (N = 9) pore formations could be detected, increasing to a maximum at 72 h (N = 10). Further incubation results in a decrease of pore detection (N = 10). Non-incubated α-syn monomers did not lead to pore detections (N = 4), as well as DMSO and Fe³⁺ (“buffer”) after all tested incubation times (N = 4, each). α-hemolysin was used as a positive control (α-HL; N = 4). <b>C</b>) In a further set of experiments, the effects of different α-syn concentrations and the effect of the aggregation inhibitor baicalein were investigated after incubation of α-syn for 72 h with DMSO and Fe³⁺. Shown is the probability of pore detection following sequential application of aliquots of the respective samples to the bilayer. α-syn was used at 7.0 µM (N = 69), 2.1 µM (N = 35) and 0.7 µM (N = 8) with up to 9 aliquots applied per sample. With decreasing α-syn concentration, cumulative pore detection rate decreases significantly (p<0.001). Co-incubation of 2.1 µM of α-syn with 50 µM of baicalein (N = 8) significantly decreases pore detection compared to 2.1 µM control condition (p<0.005). <b>D</b>) When different voltages were clamped to membranes with inserted pores, step-like changes in conductivity were consistently observed during the duration of the voltage-pulse.</p

Schematic illustration of different models for increased membrane permeability caused by α-syn oligomers.

<p>In principle, α-syn oligomers could cause increased conductance of lipid membranes by different modes of action. <b>A</b>) A diffuse damage of the bilayer could lead to an unspecific increase in transmembrane current flow. <b>B</b>) Distinct pores could be formed in the bilayer that switch between two or more different conformational states, resulting in corresponding changes in conductivity. <b>C</b>) Different numbers of uniform pores could spontaneously insert and de-insert into the membrane leading to step-like changes in conductivity. <b>D</b>) The number of “open” pores could fluctuate due to open and closure events of permanently inserted pore complexes.</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

Conformational changes measured by <i>in vitro</i> conversion reactions.

<p><b>(A)</b><i>In vitro</i> conversion reactions have been performed with radiolabeled wild-type and PrP^C(TetraH>G) purified from RK13 cells and PrP^Sc purified from brains of RML-infected Tga20 mice as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188989#pone.0188989.ref004" target="_blank">4</a>]. Samples were analyzed by SDS-PAGE-fluorography, and relative conversion efficiencies (CVE) were calculated from band intensities before and after digestion with proteinase K using the formula CVE [%] = [I°_+PK / (I°_-PK*10)]*100. PrP^C with substituted OR histidines (PrP^C(TetraH>G)) is only half as efficient in converting to the misfolded, PK-resistant conformer than wt PrP^C. Mean values ± standard error (SEM) were determined from 11 independent experiments for each PrP^C type. P-values (p (two sided) = 0.07, p (one sided) = 0.036) were obtained by T-Test calculation. (<b>B</b>) Control reactions performed in the absence of PrP^Sc seed.</p

Western blot analysis of brain lysates from RML-infected wt and transgenic mice for total PrP^C and PrP^Sc using monoclonal antibody 4H11.

<p>(<b>A</b>) Brain homogenates from terminally ill wt and PrP(TetraH>G) line 34 mice were either left untreated (- PK) or subjected to digestion with proteinase K (+ PK). Blots were reprobed for β-actin to control for equal loading. Bands corresponding to total PrP are marked on the left. Irrelevant lanes have been excised at two positions. Molecular weight standards are given on the right (in kDa). (<b>B</b>) Corresponding immunoblot analysis of brain homogenates extracted from RML-infected PrP(H95G) mice from the three different lines 11, 13, and 4, respectively, and corresponding wt control (lanes 1 and 2) before (-) and after (+) treatment with PK. Molecular weight standards are given on the right (in kDa).</p

Neuropathological changes and PrP^Sc distribution pattern in brain sections of secondary passage transgenic mice and corresponding wt controls.

<p>(<b>A</b>) First column: Paraffin-embedded tissue (PET) blots demonstrating PrP^Sc deposits in hippocampus (upper panels) and cerebellum (lower panels) of wt (C57/129Sv) and PrP(TetraH>G), line 34 inoculated with PrP(TetraH>G)-passaged prions. Second column: Corresponding PrP^Sc immunhistochemistry (mAb CDC-1). Third column: Hematoxylin and eosin stainings (H&E) of hippocampal and cerebellar sections demonstrating spongiform changes. Fourth column: GFAP immunostaining demonstrating gliosis. (<b>B</b>) Corresponding hippocampal brain sections from terminally ill PrP(H95G) mice (line 13) and corresponding wt controls (C57/129Sv) challenged with PrP(H95G)-passaged prions.</p

Lesion profiles induced by mouse-adapted scrapie isolate RML in wild-type and transgenic mice expressing mutant PrP.

<p>The extent of spongiform change (<b>A</b>) and reactive gliosis (<b>C</b>) in brain sections of terminally ill wt and PrP(TetraH>G) was assessed semi-quantitatively in a blinded fashion in nine areas of grey matter and three areas of white matter by lesion profiling. Animals were scored on a scale of 0–5 in each specific area, and mean scores (n = 6 (C57/129Sv), versus n = 7 (PrP(TetraH>G), line 34), respectively) are shown graphically (error bars plus SD). Blue diamonds: C57/129Sv. Red squares: PrP(TetraH>G). Analogous data from PrP(H95G) mice are shown in panels <b>B</b> and <b>D</b> (n = 4 (PrP(H95G), line 13), n = 5 (PrP(H95G), line 4) and n = 7 (PrP(H95G), line 11), respectively); data for C57/129Sv wt animals has been re-plotted for comparative purposes. Blue diamonds: C57/129Sv. Red squares: PrP(H95G), line 4. Green triangles: PrP(H95G), line 11. Grey circles: PrP(H95G), line 13. Scoring areas as follows: Grey matter: 1, dorsal medulla, 2, cerebellar cortex, 3, superior colliculus, 4, hypothalamus, 5, medial thalamus, 6, hippocampus, 7, septum, 8, medial cerebral cortex at septum level, 9, medial cerebral cortex at thalamus level. White matter: 1*, cerebellar white matter, 2*, mesencephalic tegmentum, 3* pyramidal tract.</p