Clusters and RNA ratios of differentially expressed genes in 3D and 2D cell cultures of A549 and UT-SCC15 cells.

<p>(A) Hierarchical clusters of genes of 2D and 3D cell cultures at day 4 after plating. Red indicates genes expressed above average; green indicates genes expressed below average and black indicates average expression after Significance Analysis of Microarrays (SAM). “Positive” indicates genes upregulated in 3D versus 2D. “Negative” indicates genes downregulated in 3D versus 2D. (B) Plot of number of transcripts against signal log ratios from 3D versus 2D cell cultures of A549 and UT-SCC15 cells. (C) Gene Ontology-dependent plotting of absolute numbers and percentage of significantly modified genes in 3D versus 2D cell cultures according to SAM analysis.</p

Validation of microarray gene expression data on protein level.

<p>(A) Signal log ratios of selected genes (FN1, CTGF, ERBB3 and BCL2A1) from DNA microarray-based analysis subjected to Western blot analysis. (B) Whole cell lysates were prepared from 3D and 2D cell cultures on day 4 after plating. SDS-PAGE and Western blot analysis were performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034279#s2" target="_blank">Material and Methods</a>. Representative images show expression of Fibronectin (240 kDa), CTGF (38 kDa), ErbB3 (180 kDa) and BCL2A1 (20 kDa). β-actin served as loading control. (C) Densitometric units of protein bands shown in ‘B’ are plotted upon normalization to β-actin.</p

Cell morphology, doubling times and cellular radiosensitivity of A549 and UT-SCC15 cells under 2D or 3D growth conditions.

<p>(A) Representative pictures and schematic of 2D (<i>i</i>, <i>iii</i>) and 3D (<i>ii, iv</i>) cell growth as found at day 4 immediately before RNA isolation. Bars, 50 µm. (B) Doubling times of 2D and 3D cell cultures at day 4 after plating. Results show mean ± SD (n = 3). n.s., not significant. Clonogenic survival of 2D or 3D grown cells exposed to increasing X-ray doses (2, 4 or 6 Gy) (C) or Cisplatin concentrations (0.1, 1, 5, 10 µM) (D) applied 24 h after plating. Cisplatin was removed after 24 h by 3- (2D) or 15-times (3D) washing with DMEM/10% FCS/1% NEAA. Results show mean ± SD (n = 3; t-test). * P<0.05; ** P<0.01. CDDP, Cisplatin. Bar, 200 µm.</p

Signal log ratios of selected genes from DNA microarray-based analysis subjected to semi-quantitative RT-PCR verification.

<p>Expression of the genes TXNIP1, DUSP6, CEACAM1, NPC1 and BCL2A1 is delineated comparatively from 3D versus 2D cell cultures of A549 and UT-SCC15 cells.</p

z-scores calculated for selected GO terms and pathways^a.

a<p>z-scores were calculated according to the GenMAPP manual based on the genes identified by t-test. A z-score value of −1.96 or 1.96 corresponds to a p-value of 0.05. The higher the absolute value of the z-score the more significant is the enrichment of changed genes in the scored pathway. Significant z-scores are high-lighted in bold letters.</p

Validation of microarray gene expression data by semi-quantitative RT-PCR.

<p>(A) Semi-quantitative RT-PCR was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034279#s2" target="_blank">Materials and Methods</a>. Shown are 1% agarose gels with RT-PCR fragments of TXNIP, DUSP6, CEACAM1, NPC1 and BCL2A1 mRNAs isolated from A549 or UT-SCC15 cells. Samples were amplified from cDNA generated by reverse transcription of total RNA of 4-day old 3D and 2D cell cultures. All experiments (V1, V2, V3) are exhibited. β-actin expression served as loading control (540 bp). (B) Graphs display results from densitometric analysis of indicated mRNA expression after normalization to β-actin. Results show mean ± SD (n = 3).</p

Distribution of raw signal intensities on all microarrays used (non-normalized)

<p><b>Copyright information:</b></p><p>Taken from "Gene expression analysis of nuclear factor I-A deficient mice indicates delayed brain maturation"</p><p>http://genomebiology.com/2007/8/5/R72</p><p>Genome Biology 2007;8(5):R72-R72.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1929142.</p><p></p> E, embryonic day; KO, knockout (); P, postnatal day; WT, wild-type ()

Distribution of normalized signal intensities on all microarrays used (per chip normalization to 50th percentile)

<p><b>Copyright information:</b></p><p>Taken from "Gene expression analysis of nuclear factor I-A deficient mice indicates delayed brain maturation"</p><p>http://genomebiology.com/2007/8/5/R72</p><p>Genome Biology 2007;8(5):R72-R72.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1929142.</p><p></p> Highly similar intensity patterns were observed for all chips used in this study, confirming that all microarray chips in these experiments are comparable. E, embryonic day; KO, knockout (); P, postnatal day; WT, wild-type ()

Murine <i>T. cruzi</i> susceptibility loci map to Chromosomes 5, 13, and 17.

<div><p>Nine microsatellite markers from the three candidate genomic regions previously identified as suceptibility loci <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000057#pone.0000057-Graefe2" target="_blank">[30]</a> were typed in an additional 22 <i>T. cruzi</i>- susceptible B6xF1 backcross mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000057#pone-0000057-t003" target="_blank">table 3</a> for respective markers).</p> <p>Single-point (dashed line) and multi-point (solid line) calculations of χ² are shown for all 68 susceptible backcross mice including the 46 from the original study.</p> <p>Dotted lines at χ² = 16.56 and χ² = 8.74 indicate thresholds for suggestive and significant linkage, respectively.</p></div

Cellular composition of the spleen from naive and <i>T. cruzi</i>- infected mice.

<div><p>Single spleen cell suspensions from naive mice (A) and from mice infected on day 24 with <i>T. cruzi</i> (B) were analysed by flow cytometry.</p> <p>Cells were stained for both CD4 and CD8, for CD19, for CD11b, or with propidium iodide (PI) and Annexin V (AxV).</p> <p>Numbers indicate percentage of cells expressing the respective marker.</p> <p>B6, C57BL/6 mice; F1, B6D2F1 mice.</p></div