Network of differential level proteins in cholesteatoma with associations to connective tissue.

<p>A. The top scoring, automatically-synthesized network of related proteins in IPA: Connective Tissue Development and Function, Embryonic Development, Organ Developmentive tissue.d proteins: Group B proteins.d EACS.holesteatoma a STRING. All proteins, except for MMP9, showed the same expression direction comparing cholesteatoma with tympanic membrane and EACS, respectively. Protein level differences meeting the group A or B criteria were detected in at least one of the two comparisons. 41 interactions (7.95 expected) were identified between the 23 proteins, network <i>p</i> value = 1.11e-16. B. Some of the top scoring significant associations of the network with: GO Biological Processes, GO Cellular Components, and KEGG Pathways; ordered by <i>p</i> value.</p

Activation direction of biological functions in cholesteatoma.

<p>Statistical predictions on the direction of activation of biological functions associated with cholesteatoma (see experimental procedures). Biological functions with z - scores of the predictions above the numerical value 1.645 (90% significance level) are shown. In a first step, biological functions were statistically associated with cholesteatoma based on the up- and down-regulated proteins that could be assigned to these functions. Subsequently, predictions on the activation direction were calculated from the composition of up- and down-regulations among these proteins. The listed results were found in comparisons between cholesteatoma sack (C), neck of cholesteatoma (N), tympanic membrane (T), and external auditory canal skin (S). *The proteins had to show the same direction of expression in both tissues and meet the fold change criteria in at least one of the tissues.</p

Top 20 Up-regulated proteins in cholesteatoma.

<p>Up-regulated proteins in cholesteatoma sorted by 1: The number of tissues compared to which cholesteatoma showed higher levels of the protein; 2: Protein group (fold change criteria A then B); 3: Fold change. C: Cholesteatoma; N: Neck of cholesteatoma; T: Tympanic membrane; S: External auditory canal skin; a: Protein group A; b: Protein group B; b(t): Triplicates in protein group B.</p

Overview of levels of 11 related proteins involved in inflammation, response to bacteria, and/or protein degradation.

<p>Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues. Left: Leukocyte-associated proteins. Middle: Inhibitor of enzyme activity. Right other protein degrading and/or immune-response related proteins. a: Group A proteins, b: Group B proteins, b(t): Triplicate values in group B. Gene names below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Simple overview of workflow.

<p>EACS: external auditory canal skin; Tymp: tympanic membrane; Neck: neck of cholesteatoma; Chol: cholesteatoma; Muc: middle ear mucosa; ME: Middle ear; LC: Liquid Chromatography; MS: Mass Spectrometry.</p

Cytokeratins.

<p>All identified cytokeratins (Hair- and hair follicle keratins excluded) were grouped according to Moll <i>et al</i>.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104103#pone.0104103-Moll1" target="_blank">[38]</a>. Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues (results from comparisons including the middle ear mucosa are not shown). a: Group A proteins; b: Group B proteins; b(t): Triplicate values in group B. Gene names shown below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Minimum fold changes of proteins meeting the group A and B criteria for fold change.

<p>Only comparisons including cholesteatoma are shown. Neck: Neck of cholesteatoma; Tymp: Tympanic membrane; EACS: external auditory canal skin; Mucosa: Middle ear mucosa.</p

Synthesized network of immune response-related up-regulated proteins in cholesteatoma and neck of cholesteatoma.

<p>A. All proteins showed higher protein levels in cholesteatoma and neck of cholesteatoma compared to the external auditory canal skin. The proteins met the fold change criteria in at least one of the two tissue comparisons. B. Examples of significant associations of the network with: GO Biological Processes, GO Cellular Components and KEGG Pathways; ordered by <i>p</i> value. The networks were generated in STRING.</p

Identified proteins.

<p>Number and overlap of unique proteins identified in the three separate mass spectrometry experiments. In each experiment, a sample set of the five tissue types (pooled samples from three patients) were analyzed.</p

Canonical pathways associated with the proteins meeting the fold-change criteria.

<p>A. The top scoring (lowest <i>p</i>-values) canonical pathways associated with the differentially expressed proteins found in the pairwise comparisons between the four keratinizing tissues. *The second highest score "Methylglyoxal Degradation III" was found in the comparison between tympanic membrane and EACS. All others were found in the comparison between cholesteatoma and EACS. Horizontal blue line indicates <i>p</i> value  = 0.05. B. <i>p</i> values and the involved differential-level proteins of three selected pathways from the comparison between cholesteatoma and EACS. All proteins showed lower levels (green color) in cholesteatoma compared with EACS. Underlined proteins: Group B proteins.</p