Altered proliferation in the skin of <i>Miz1ΔPOZ</i> mice depends on p21^Cip1.

<p>TPA treatment of skin from control (<i>Ctr</i>) and <i>Miz1</i>Δ<i>POZ</i> mice on a <i>cdkn1a</i> null background shows the same increase of the Ki67 labelling index and scattering of Ki67 positive cells in the suprabasal layers of the epidermis (<b>A–D</b> and <b>F</b>; n = 3 for each genotype and condition; <i>Ctr</i> vs <i>Miz1</i>Δ<i>POZ</i> for −TPA: p = 0.4007; <i>Ctr</i> vs <i>Miz1</i>Δ<i>POZ</i> for +TPA: p = 0.4494). In contrast, the equivalent experiment on a <i>cdkn2b</i> null background (<b>E</b>; n = 3 for each genotype and condition) exhibited the same result as in <i>cdkn2b^+/+</i> animals (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034885#pone-0034885-g002" target="_blank">Figure 2 M</a>; <i>Ctr</i> vs <i>Miz1</i>Δ<i>POZ</i> for ±TPA: p<0.0001). In <i>cdkn1a^+/+</i> animals, p21^Cip1 is upregulated in protein extracts from <i>Miz1</i>Δ<i>POZ</i>-mice (<i>ΔPOZ</i>) compared to extracts from control-mice (<i>Ctr</i>) either with or without TPA-treatment (<b>I</b>). Sciatic nerve extracts from <i>cdkn1a</i>^+/+ and <i>cdkn1a</i>^−/− animals were used as positive and negative controls, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034885#s4" target="_blank">Material and Methods</a>). Chromatin immunoprecipitation assay using chromatin from murine PAM212 keratinocytes and antibodies against Miz1 and Myc (<b>G</b>; <b>H</b>; one representative experiment of three independent experiments each). The experiment demonstrates that both transcription factors bind to the <i>cdkn1a</i> promoter in this cell type. Miz1 binds to the <i>cdkn1a</i> promoter in PAM212 keratinocytes, without and with TPA treatment (100 nM for 4 hours). Primers used either amplified genomic DNA comprising part of the <i>cdkn1a</i> promoter (p21) or a <i>cdkna1a</i> unrelated sequence of chromosome 17 (ctr). For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034885#s4" target="_blank">Material and Methods</a>. The graphs show the mean value of 2–3 technical replicas.