Expression of chemokines.

<p><b>A.</b> SQRT-PCR of chemokine mRNA expression in NEB-1, KEB-7 and EBDM-1 (n = 3 to n = 5). <i>CXCL1</i>, <i>CXCL8/IL-8</i> and <i>CXCL14</i> expression was increased in KEB-7. Only <i>CXCL11</i> and <i>CXCL14</i> were increased in EBDM-1. <b>B.</b> CXCL8/IL-8 ELISA of 48-h-conditioned cell culture supernatant showed 2-fold upregulation at the protein level in KEB-7 but not in EBDM-1, which correlates with the SQRT-PCR results (n = 4). <b>C.</b> CXCL8/IL-8 concentrations were highly increased in EBS patients blister fluids. In blister fluids of healthy controls no CXCL8/IL-8 was detectable (n = 3 to n = 4). The numbers correlate with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070123#pone-0070123-t001" target="_blank">Table 1</a>. Student’s <i>t</i>-test was performed with p values: * ≤0.05, *** ≤0.005, Δ≤0.001, ‡ = no significant difference between investigated cell lines. (In 6C, the Student’s <i>t</i>-test compared the entire patient group to the entire control group).</p

Type I and type II cytokeratin expression.

<p><b>A.</b> SQRT-PCR shows an increase in mRNA expression of type I cytokeratins <i>K14</i>, <i>15</i>, <i>16</i> and <i>17</i> in KEB-7 (K14 R125P) and EBDM-1 (K14 R125H) cell lines compared to NEB-1 wild-type keratinocytes (n = 4 to n = 7). <b>B.</b> SQRT-PCR of type II cytokeratins shows only a significant increase of <i>K5</i> mRNA expression in the KEB-7 cell line (n = 3 to n = 5). <b>C.</b> Western blot analysis from whole-cell lysates revealed increased protein expression of K14, 15 and 16 in both EBS-DM cell lines. Annexin-I was used as a loading control. Student’s <i>t</i>-test was performed with p values: * ≤0.05, ** ≤0.01, Δ≤0.001, ΔΔ≤0.0005, ΔΔΔ≤0.0001, ‡ = no significant difference between investigated cell lines.</p

Aberrant regulation of actin cytoskeleton components.

<p><b>A.</b> SQRT-PCR shows increased <i>WIPF1</i> mRNA expression in KEB-7 and EBDM-1 cell lines compared to NEB-1 wild-type keratinocytes (n = 3). <b>B.</b> SQRT-PCR shows increased <i>ARHGEF9</i> mRNA expression in KEB-7 and EBDM-1 cell lines compared to NEB-1 wild-type keratinocytes (n = 5). <b>C.</b> Immunoprecipitation and western blot show increased amounts of active (GTP-bound) Cdc42 protein in KEB-7 and EBDM-1. Annexin-I was used as a loading control. <b>D.</b> Western blot of whole-cell lysates shows increased amounts of phospho-ERM proteins in KEB-7 and EBDM-1. Annexin-I was used as a loading control. Student’s <i>t</i>-test was performed with p values: * ≤0.05, ** ≤0.01, ΔΔ ≤0.0005.</p

Microarray analysis of kallikrein-related peptidases.

<p>Microarray analysis of kallikrein-related peptidases.</p

Expression of matrix metalloproteinases.

<p><b>A.</b> SQRT-PCR shows increased matrix metalloproteinase mRNA expression in EBS-DM cell lines (n = 4). <b>B.</b> MMP-9 ELISA of 48-h-conditioned cell culture supernatant shows 2-fold upregulation of MMP-9 in KEB-7 and 46-fold upregulation in EBDM-1 at the protein level (n = 4). <b>C.</b> MMP-9 levels were highly increased in EBS patients blister fluids compared to healthy controls (n = 3 to n = 4). The numbers correlate with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070123#pone-0070123-t001" target="_blank">Table 1</a>. Student`s <i>t</i>-test was performed with p values: * ≤0.05, ** ≤0.01, *** ≤0.005, ΔΔ≤0.0005, ΔΔΔ≤0.0001. (In 2C, the Student’s <i>t</i>-test compared the entire patient group to the entire control group).</p

Schematic representation of pathways involved in EBS-DM pathomechanisms.

<p><b>A.</b> IL-1β activates the JNK pathway in Dowling-Meara cell lines and induces expression of target genes. The IL-1 pathway also interacts with the Cdc42 pathway. <b>B.</b> Schematic representation of the Cdc42 pathway. Upregulation of collybistin (ARHGEF9) leads to increased GDP-GTP exchange and activation of Cdc42. Active Cdc42 activates its downstream effectors like WASP (<u>W</u>iskott-<u>A</u>ldrich <u>S</u>yndrome <u>P</u>rotein) and Arp2/3 (<u>A</u>ctin-<u>r</u>elated <u>p</u>rotein 2 and 3) as well as MRCK and ERM proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070123#pone.0070123-Kedrin1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070123#pone.0070123-Kurisu1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070123#pone.0070123-Mullins1" target="_blank">[43]</a>. The Arp2/3 complex serves as a nucleation core for actin polymerization, and phospho-ERM proteins connect actin filaments to the plasma membrane. <i>WIPF1</i> expression was also upregulated in EBS-DM cell lines. The Cdc42 downstream-effects contribute to actin dynamics, invasiveness and, through PAK, to the gene expression levels. <b>C.</b> Depiction of the interconnected network of small GTPases like Rho, Rac and Cdc42 as well as CXCL8/IL-8. Activated Cdc42 leads to increased amounts of phospho-ezrin. After activation, p-ezrin recruits the GEF Dbl to lipid rafts and induces activation of both Cdc42 in a feedback loop as well as the Rho pathway. Binding of CXCL8/IL-8 also leads to activation of Rac1/RhoA and Cdc42, also resulting in expression of target genes. (Red colored items indicate aspects investigated in the present study).</p

Microarray analysis of genes involved in actin cytoskeleton dynamics.

<p>Microarray analysis of genes involved in actin cytoskeleton dynamics.</p

Microarray analysis of cytokeratin genes.

<p>Microarray analysis of cytokeratin genes.</p

Expression of junction proteins in EBS-DM cell lines.

<p><b>A.</b> SQRT-PCR of desmocollin mRNA expression in NEB-1, KEB-7 and EBDM-1 (n = 3 to n = 4). <i>DSC1</i> shows the highest increase in both EBS-DM cell lines. <i>DSC2</i> and <i>DSC3</i> are increased only in EBDM-1 but not in KEB-7. <b>B.</b> SQRT-PCR of desmoglein mRNA expression in NEB-1, KEB-7 and EBDM-1 (n = 3 to n = 5). <i>DSG1</i> shows the highest increase in EBDM-1. Only a slight increase was observed for <i>DSG3</i>. <i>DSG4</i> is only significantly increased in KEB-7. <b>C.</b> SQRT-PCR of gap junction protein mRNA expression in NEB-1, KEB-7 and EBDM-1 (n = 3 to n = 5). <i>GJA1</i> and <i>GJB6</i> expression is increased in both EBS-DM cell lines. <i>GJB2</i> is only increased significantly in KEB-7. Student’s <i>t</i>-test was performed with p values: * ≤0.05, ** ≤0.01, *** ≤0.005, ‡ = no significant difference between investigated cell lines.</p