Overlay of three mass spectra showing the ΔF508-specific mass signal changes generated in the T-specific cleavage reaction of the reverse strands

<p><b>Copyright information:</b></p><p>Taken from "Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS"</p><p>Nucleic Acids Research 2005;33(4):e38-e38.</p><p>Published online 24 Feb 2005</p><p>PMCID:PMC549577.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The mass signal pattern is derived from a triplexed reaction encompassing exon 10, 21 and 24 of the CFTR gene. Three different individuals were analyzed, the upper mass spectrum representing a homozygous wild-type for ΔF508, a carrier of the ΔF508 mutation (middle) and an individual homoyzgous mutant ΔF508 (lower mass spectrum). Mass signals specific for the mutation are indicated with a line

Gradient PAGE gel with CYBR Gold staining showing the DNA fragmentation of untreated genomic DNA (left) and after bisulfite treatment at varying temperatures (from left to right: 50, 70 and 80°C)

<p><b>Copyright information:</b></p><p>Taken from "A new method for accurate assessment of DNA quality after bisulfite treatment"</p><p></p><p>Nucleic Acids Research 2007;35(5):e29-e29.</p><p>Published online 26 Jan 2007</p><p>PMCID:PMC1865059.</p><p>© 2007 The Author(s).</p> The figure indicates that an increase of the incubation temperature during bisulfite treatment results in increased DNA fragmentation

Panel () shows the probability distributions for observed methylation ratios based on the binomial distribution and different amounts of starting molecules

<p><b>Copyright information:</b></p><p>Taken from "A new method for accurate assessment of DNA quality after bisulfite treatment"</p><p></p><p>Nucleic Acids Research 2007;35(5):e29-e29.</p><p>Published online 26 Jan 2007</p><p>PMCID:PMC1865059.</p><p>© 2007 The Author(s).</p> Shown are examples for 10, 25, 50 75 and 90% methylated molecules in the starting template. With a sample size of 3000 molecules, 95% of all randomly sampled probes will contain between 48 and 52% methylated DNA when the DNA sample contains 50% methylated DNA (red colored distribution). However, when the DNA sample contains only 300 molecules, this range is expanded from 43 to 57% (blue colored distribution). Panel () shows the 95% confidence intervals for sampling-means as a function of the number of the sampled molecules. Shown are results for 10 (blue), 25 (red) and 50% (black) methylated molecules in the starting template

Box plot graphic depicting the variability of repeated measurements for each step in the process (Step 1: bisulphite treatment; Step 2: PCR; Step 3: MassCLEAVE; Step 4: MALDI-TOF MS analysis)

<p><b>Copyright information:</b></p><p>Taken from "A new method for accurate assessment of DNA quality after bisulfite treatment"</p><p></p><p>Nucleic Acids Research 2007;35(5):e29-e29.</p><p>Published online 26 Jan 2007</p><p>PMCID:PMC1865059.</p><p>© 2007 The Author(s).</p> Boxes are centered on the median and range from the lower to the upper quartile. Whiskers indicate the interquartile range. Red whiskers indicate the standard deviation from the mean. Bisulfite treatment and PCR can be identified as the greatest source of process variability. The post-PCR processing (MassCLEAVE) and, in particular, the MALDI analysis show high precision in repeated measurements

Correlation between the results obtained from the quality control assays and PCR success from additional genomic targets of varying length

<p><b>Copyright information:</b></p><p>Taken from "A new method for accurate assessment of DNA quality after bisulfite treatment"</p><p></p><p>Nucleic Acids Research 2007;35(5):e29-e29.</p><p>Published online 26 Jan 2007</p><p>PMCID:PMC1865059.</p><p>© 2007 The Author(s).</p> The bar graphs in panel () and () show the results from the quality control assays similar to Figure 5. The QC assay indicates that incubation at 90°C limits amplification to only short amplicons

Characterization of Sox2 translation during OCSC isolation.

<p>(<b>A</b>,<b>B</b>) Double-labeling of Sox2 with PCNA, Bmi1, Jag1 and Hes1 in the OC at E13.5 and P4 compared to OCSCs. (<b>A</b>) Representative immunostaining images of longitudinal cryosections of the prosensory domain in the proximal cochlea duct at E13.5 (basilar membrane on top, luminal surface on the bottom). (<b>B</b>) Immature (P4) OC in mid-modiolar sections of the basal cochlea turn (medial to the left). (<b>C</b>) P4 OC-derived otic spheres after 5 DIV. Due to the requirements for the different tissue types investigated, the fixation, staining protocols and image acquisition settings were not identical (Scale Bars: A,B,C, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s005" target="_blank">Figure S5</a>).</p

Epigenetic and transcriptional characterization of Sox2 during OCSC isolation.

<p>(<b>A</b>,<b>B</b>,<b>C</b>) Methylation profile of the Sox2 enhancers (<b>A</b>) SRR1/2, (<b>B</b>) NOP1 and (<b>C</b>) NOP2 in OCSCs as compared to the OC at P4 and E13.5 (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>D</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) in OCSCs and the OC at E13.5 and P4. Relative expression levels of OCSCs and E13.5 OC were compared with those of P4 OC. Transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of three independent experiments with SDs are depicted (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2C</a>).</p

Differentiation potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) Methylation profiles of the otic Sox2 enhancers (<b>A</b>) NOP1 and (<b>B</b>) NOP2 in the mature OC (P21), proliferating OCSC spheres and epithelial patches differentiated from OCSC spheres (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>C</b>) Relative expression levels of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) after 14 and 28 days of differentiation (n = 3) were compared with those of the proliferating OCSC spheres by qPCR. Transcript levels were normalized to TbP/Ubiquitin C levels. Shown are averages of three independent experiments (and two independent experiments for 28 days for the differentiation group) with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2D</a>). (<b>D</b>–<b>G</b>) <i>In situ</i> cell type-specific marker expression of the maturing OC (P4): Sox2 antibody (<b>F</b>) labels all supporting cells of the sensory domain (<b>G</b>), whereas S100-antibody (<b>D</b>) detects pillar and Deiters' cells only (<b>G</b>). Myosin VIIa (<b>E</b>) expression is associated with inner and outer hair cells (<b>G</b>). (<b>H</b>–<b>K</b>) OCSC-derived progeny differentiated under <i>in vitro</i> culture conditions. OCSC progeny were labeled by an EdU pulse (during the last day of 5 DIV) under proliferative culture conditions and a pulse chase after 14 DIV under differentiation-inducing culture conditions. EdU-labeling in supporting cell (Sox2, S100) (<b>H</b>) and hair cell-like (myosin VIIa) (<b>I</b>) cells. Hair cell-like cells were additionally characterized based on membrane-localized prestin (<b>J</b>) and F-actin-stained (<b>K</b>) membrane protrusions (Scale Bars: D,E,F,H,I,J,K, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s006" target="_blank">Figure S6</a>).</p

EGF interferes with the epigenetic regulation of Sox2 expression and affects the self-renewal potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) P4 OC-derived otospheres after 5 DIV; labeling for Sox2 combined with EdU and DAPI (<b>A</b>) Otospheres grown under FGF/IGF-only conditions. (<b>B</b>) Otospheres supplemented with EGF as an additional growth factor (Scale Bars: A,B, 100 µm). (<b>C</b>) Absolute numbers of primary spheres isolated per OC with (n = 7) and without EGF (n = 8) supplementation. Data were analyzed by student's t-test and are presented as means ±SDs. (<b>D</b>) Mean diameter of the primary sphere population measured in a range from 25 to 60 µm with (n = 7) and without EGF (n = 8) supplementation. Data are presented as means ±SDs. (<b>E</b>) Methylation profiles of the otic Sox2 enhancers NOP1/2 in P21 OC, proliferating OCSCs and OCSCs supplemented with EGF (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>F</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin VIIa, p27Kip and Prox1) in standard OCSCs and in OCSCs supplemented with EGF. Relative expression levels of standard OCSCs were compared to those of OCSCs supplemented with EGF. Transcript levels were normalized to HPRT1/TbP levels. Averages of three independent experiments are shown with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2E</a>).</p

Epigenetic, transcriptional and translational characterization of Sox2 expression during OC development.

<p>(<b>A</b>–<b>C</b>) OC during development. (<b>A</b>) Upper panel: Schematic of the sensory domain, which contains the proximal cochlea duct, showing interkinetic nuclear migration at E13.5. Sox2 expression is indicated by red nuclei. Remaining panels: marker expression at E13.5. All proliferating Ki-67-positive cells are co-labeled for Sox2. (<b>B</b>) Upper panel: schematic of the different cell types found in the maturating OC at P4. Inner hair cell (ihc, arrowhead), three outer hair cells (ohc, arrowheads) and different supporting cells: inner sulcus cells (is); interphalangeal cells (i); pillar cells (p); Deiters' cells (d); Hensen's cells (h); and Claudius cells (c). Remaining panels: marker expression at P4. The quiescence of Sox2-positive supporting cells is indicated by co-labeling with p27Kip1. (<b>C</b>) Upper panel: schematic of the different cell types found in the functional OC at P21. Remaining panels: marker expression at P21. Senescence of Sox2-positive cells is indicated by p16Ink4a expression. (<b>D</b>) RT-PCR of pluripotency marker, hair cell marker and supporting cell marker expression in the OC (E13.5, P4, P21). HPRT1 was used as the loading control. (<b>E</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, Myosin VIIa, p27Kip1 and Prox1) during OC development (E13.5, P4 and P21). The relative expression levels of P4 and P21 were compared with those at E13.5. The transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of the three independent experiments with SDs are shown (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2B</a>). Depending on the temporal expression pattern, genes were assigned to early, transition or differentiation groups (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s003" target="_blank">Figure S3</a>). (<b>F</b>,<b>G</b>) Bisulfite methylation of the Sox2 enhancers (f) (NOP1/2) and (g) (SRR1/2) during OC development (E13.5, P4, P21) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (Scale Bars: A,B,C, 10 µm).</p