NEMO knockout murine dermal fibroblasts show a decreased nuclear translocation of p65.

<p><b>a.</b> MTT assay determined optimal experimental conditions. 80% viable cells was set as threshold. After overnight serum starvation MDFs were treated with etoposide for 3 h followed by a 24 h incubation period. MTT assay was started afterwards to determine the viability of cells. Values are presented as mean ± SEM in percent. (<i>n</i> = 3) <b>b.</b> In order to evaluate DNA damage response and cell cycle arrest mRNA expression of p21 was analysed by RT-qPCR in MDFs treated with 25μM etoposide for 3 h followed by a 24 h incubation time (<i>n</i> = 5). Values are presented as mean ± SEM of fold change. Comparison was made with two-tailed t-test; <i>P</i>-value indicated the significance of difference. <b>c.</b> Representative immunostaining of γH2Ax (green) and p65 (red) in wildtype (NEMO WT) and NEMO knockout (NEMO k/o) MDFs treated with 25μM etoposide for 3 h with a following incubation period of 24 h. Scale bars, 50μM. The graph shows the percentage of p65 in the cytoplasm (black bars) compared to the nucleus (grey bars) as percentage of red pixels. Values are mean ± SEM in percent. Comparison was made with two-tailed t-test (<i>n</i> = 10); line and <i>P</i>-value.</p

Schematic overview of the experimental workflow.

<p>Murine dermal fibroblasts (MDFs) are isolated from NEMO-floxed mice. After short expansion in cell culture these MDFs are transfected with pCAG-Cre-T2A-mRuby2 or pCAG-mRuby2, respectively. Because of mRuby2 expression, successfully transfected cells can be sorted by FACS. Cells transfected with pCAG-Cre-T2A-mRuby2 are knocked out for NEMO while pCAG-mRuby2 transfected cells are used as wildtype controls. After transfection cells are treated with 25 μM etoposide for 3 h to induce DNA damage. 24 h after treatment cell culture media is taken for ELISA measurement of secretion and cells are harvested for RNA isolation and subsequent RT-qPCR analysis.</p

Boolean network for gene regulation during cell cycle progression and the onset of cell cycle arrest after DNA damage.

<p>Boolean Rules using operators “&” (logical and), “|” (logical or) and “¬” (logical not).</p

Knockouts that cause <i>in-silico</i> IL-6 and IL-8 inhibition for NEMO knockout.

<p>NEMO is switched off (NEMO = 0) preventing NF-κB signaling from being activated. The outcome is similar to the two previously described simulations in Figs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005741#pcbi.1005741.g004" target="_blank">4</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005741#pcbi.1005741.g005" target="_blank">5</a>.</p

DNA damaged NEMO knockout MDFs show a decrease in IL-6 and IL-8 mRNA expression and protein secretion.

<p><b>a</b>. To assess the influence of the NEMO knockout on DNA damage mediated activation of SASP signaling IL-6 mRNA expression was measured by RT-qPCR in untreated and etoposide-treated MDFs (<i>n</i> = 5). Cells with wildtype NEMO (black bars) or NEMO knockout (grey bars) were used. Values were presented as mean ± SEM of fold change. Comparison was made with the two-tailed t-test. <b>b</b>. IL-6 secretion was measured by ELISA in conditioned media of untreated and etoposide-treated MDFs (<i>n</i> = 5). Cells with wildtype NEMO (black bars) or NEMO knockout (grey bars) were used. Values were presented as mean ± SEM of total secretion in pg/ml, nd means non-detectable. Comparison was made with the two-tailed t-test. <b>c</b>. In addition to IL-6 murine IL-8 homologues KC, LIX and MIP-2 were used to further show activation of SASP signaling. mRNA of all three homologues was measured by RT-qPCR in untreated and etoposide-treated MDFs (n = 5). Cells with wildtype NEMO (black bars) or NEMO knockout (grey bars) were used. Values were presented as mean ± SEM of fold change. Comparison was made with the two-tailed t-test. <b>d</b>. IL-8 homologue secretion was measured by ELISA in conditioned media as previously described (n = 5). Values were presented as mean ± SEM of total secretion in pg/ml, nd means non-detectable. Comparison was made with the two-tailed t-test.</p

Knockouts that cause <i>in-silico</i> IL-6 and IL-8 inhibition for IkB overexpression.

<p>This simulation shows an overexpression of IκB (IkB = 1) showing a similar outcome as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005741#pcbi.1005741.g004" target="_blank">Fig 4</a>.</p

Knockouts that cause <i>in-silico</i> IL-6 and IL-8 inhibition for NFkB knockout.

<p>Network states present the gene activity of all genes in the model. Green boxes indicate gene activation while red boxes show gene inactivation. A knock-down or overexpression is simulated by setting a gene to 0 or 1, respectively. This simulation shows the time course of expected states after DNA damage with NF-κB switched off (NFkB = 0) which leads to an inhibition of proinflammatory signaling.</p

Boolean network for gene regulation during cell cycle progression and the onset of cell cycle arrest after DNA damage.

<p>The overview shows the network wiring of the known gene regulations during DNA damage with a focus on the DNA damage repair/cell cycle arrest signaling. Cell cycle arrested cells over time show a tendency to develop a secretory phenotype that causes them to secrete high amounts of proinflammatory factors that can negatively influence neighboring cells. Major signaling pathways of these factors are included in this overview and in the Boolean network. Arrows indicate gene activation and inhibition is depicted as bar head. However, the interaction may be more complex and the corresponding Boolean rules are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005741#pcbi.1005741.t001" target="_blank">Table 1</a>.</p

Naturally occurring network states.

<p>Without DNA damage the resulting network state is expected to show normal cell cycle progression. As shown here this includes the activation of CDK2 (t = 5) and CDK4 (t = 2) with a subsequent phosphorylation of RB (t = 3) leading to a release of E2F (t = 4) which will release the cell into cell cycle progression. The temporal sequence is shown as t = n. Active genes are shown as green, inactive genes as dark purple.</p

NEMO knockout murine dermal fibroblasts show a decreased nuclear translocation of p65.

<p><b>a.</b> MTT assay determined optimal experimental conditions. 80% viable cells was set as threshold. After overnight serum starvation MDFs were treated with etoposide for 3 h followed by a 24 h incubation period. MTT assay was started afterwards to determine the viability of cells. Values are presented as mean ± SEM in percent. (<i>n</i> = 3) <b>b.</b> In order to evaluate DNA damage response and cell cycle arrest mRNA expression of p21 was analysed by RT-qPCR in MDFs treated with 25μM etoposide for 3 h followed by a 24 h incubation time (<i>n</i> = 5). Values are presented as mean ± SEM of fold change. Comparison was made with two-tailed t-test; <i>P</i>-value indicated the significance of difference. <b>c.</b> Representative immunostaining of γH2Ax (green) and p65 (red) in wildtype (NEMO WT) and NEMO knockout (NEMO k/o) MDFs treated with 25μM etoposide for 3 h with a following incubation period of 24 h. Scale bars, 50μM. The graph shows the percentage of p65 in the cytoplasm (black bars) compared to the nucleus (grey bars) as percentage of red pixels. Values are mean ± SEM in percent. Comparison was made with two-tailed t-test (<i>n</i> = 10); line and <i>P</i>-value.</p