Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-3

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>ichment of target genes relative to IgG on an agarose gel. (B) Real-time PCR demonstrating the relative enrichment of p63 target genes with p63 antibodies and IgG. Primer sequences utilized are listed in Additional File

Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-5

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>omoter demonstrate high levels of activity when co-transfected with an expression plasmid encoding ΔNp63α in PtK2 cells. Luciferase values were determined and normalized against β-galactosidase values. Corrected luciferase values for cells transfected with empty vector pCMV-HA were set at 1. TK; thymidine kinas

Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-1

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>s well as at the 5' end and 3' end of these target genes

Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-2

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>in Additional File to specifically amplify the target genes. β-Actin serves as a control

Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-6

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>ck-transfected cells were used as a control. Primer sequences used are listed in Additional File

<i>S. cerevisiae</i> forms filamentous mats.

<p><b>A</b>) Wild-type cells (PC313) were spotted 2 cm apart onto 0.3% agar media that contained (YEPD; top panel) or lacked (YEP; bottom panel) glucose. The YEPD plate was incubated for 4 days and photographed; the YEP plate for 15 days. Bar = 1 cm. <b>B</b>) Microscopic examination of perimeters of mats in 1A. Bar = 100 microns. <b>C</b>) The origin of filamentous mats. Wild type (PC538) cells were examined on synthetic medium either containing 2% glucose (SCD) or lacking glucose (SC) in 0.3% agar medium for 24 h at 30°C. A compiled Z-stack rendering of typical microcolonies are shown. Bar = 20 microns. <b>D</b>) Same strains in 1C were examined on rich medium either containing 2% glucose (YEPD) or lacking glucose (YEP) in 0.3% agar. A representative microscopic image is shown. Bar = 10 microns. E) Vegetative mats mature into filamentous mats over time as nutrients become limiting. Two mats of wild type (PC313) strain were spotted bilaterally (1.5 cm apart) on YEPD and YEP media (+0.2% galactose) containing 0.3% agar media. The number of filaments occurring along the circumference of mats was scored on a scale of 1, 2, or 3 dots at 20× magnification corresponding to 3, 6, or 9 filaments or greater, respectively. Dots were plotted on a circle representing the outline of one of the mats with right hemispheres corresponding to the side of the mat facing a second mat. Asymmetric filamentation observed in the right hemisphere of 2d, Glu can possibly result from nutritional stress compounded by nutrient depletion from adjacent mats. Filamentation was monitored and plotted after growth for 1, 2, 3, and 4 days. Quantitation of pseudohyphae was complicated at longer time points when biofilms began to variegate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Halme1" target="_blank">[60]</a>. Bar = 1 cm.</p

MAPK- and Flo11-dependent colony avoidance response.

<p><b>A</b>) Wild-type cells were spotted in three spots and examined daily. The photograph showing the embossed appearance of colonies was taken at day 3. Left three panels, Bar = 1 cm. Far right panel, micrograph of cells at the perimeter of an asymmetrically forming biofilm. Bar, 200 microns. Mat borders facing (red arrows) or not facing (blue arrows) another mat are indicated. <b>B</b>) Wild type (PC538), <i>flo</i>11Δ (PC1029), and <i>ste</i>12Δ (PC2382) cells were grown on YEPD media for 18 h. Equal concentrations of cells were spotted, 1 cm apart, on to YEPD media containing 0.3% agar. Plates were incubated for 48 h at 30°C and photographed using transmitted light. Bar = 1 cm. <b>C</b>) Bar graph of height measurements (in mm) of the mat borders facing/not facing the adjacent mats on the right in B. Contour maps in the Z-axis of mats was generated. Seven readings after the first peak in the Z-axis were averaged to plot the graph. Standard deviation between measurements were used to generate the error bars.</p

Model for the different mat responses controlled by the MAPK pathway.

<p>Schematic of a mat expanding under nutrient-limiting conditions is shown. Different responses regulated by the MAPK pathway may include: 1) mat expansion in the plane of the XY-axis (surface growth, through Flo11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Reynolds1" target="_blank">[23]</a>), 2) cell differentiation that causes invasive growth in the Z-axis (downward growth, Flo11 and differentiation), and 3) upward growth in the plane of the Z-axis in response to surface rigidity and nutrient-limiting conditions (Flo11 and differentiation). This upward growth may represent a type of chemorepulsion. An extracellular matrix (ECM), which may contain shed Flo11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Karunanithi1" target="_blank">[48]</a> as well as other proteins is depicted.</p

The role of Flo11 overexpression on upward growth in the plane of the Z-axis.

<p><b>A</b>) Microcolonies of wild-type cells (PC538) and cells overexpressing <i>FLO11</i> (PC2712) were examined by microscopy at 10× after 24 h incubation at 30°C. Wild type and <i>ste12</i> mats on high agar concentrations is also shown Bar = 100 microns. <b>B</b>) Contour mapping of z-stack rendering of the indicated microcolonies in panel 7A are shown. Bar = 30 microns. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s006" target="_blank">Supplemental Movies S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s007" target="_blank">S6</a>. <b>C</b>) Wild-type (PC538), <i>flo11</i>Δ (PC1029), and <i>GAL-FLO11</i> (PC2712) cells were spotted onto YEP-GAL medium (8%) agar atop nitrocellulose filters for 24 h at 30°C. Colonies were photographed in transmitted light. Bar = 1 cm. At right, separation of the <i>GAL-FLO11</i> mat from the surface using forceps. <b>D</b>) Microscopic examination of the mats in panel C. Bar = 200 microns.</p

The role of the MAPK pathway in regulating mat architecture when exposed to surfaces of different rigidities.

<p><b>A</b>) Contour maps in the Z-axis of wild type (PC538) mats incubated in media of different agar concentrations for 14d. Insets show mat morphology (left, photograph, bar, 1 cm; right, photomicrograph, bar, 200 microns) in 4% agar. The numbers in parentheses represent the average mat dry weight from two experiments with standard deviation shown. Scale bars for the X and Y-axes are in mm. <b>B</b>) Mats formed by a <i>ste12</i>Δ mutant (PC539) on different agar concentrations. Analysis is as described for panel A.</p