<i>Wwox-KO BK5-Cre</i> transgenic mice model gene expression profile study.

<p>(A) Heatmap of the differentially expressed genes between <i>Wwox WT vs. Wwox KO</i> mammary gland epithelial organoid samples (p<0.01; 2 Fold changes). Color scale at bottom of picture is used to represent expression level: low expression is represented by green, and high expression is represented by red. (B) Expression graph of the 913 deregulated probes (136 probes down-modulated and 777 up-modulated) among <i>Wwox WT</i> and <i>Wwox KO</i> mammary epithelial organoid samples. (C) Scatterplot graph showing the representative clusters, after redundancy reduction of the statistical significant GO terms (p<0.025) enriched in the deregulated gene list, in a two dimensional space related to GO terms' semantic similarities. Bubble color indicates the p-value of GO terms (expressed as Log10 p-value) and bubble size indicates the frequency of the GO term in the underlying GOA database (bubbles of more general terms are larger).</p

<i>Wnt5a</i> expression increases in <i>BK5 Wwox KO</i> mammary epithelium.

<p>Semi-quantitative RT-PCR was performed on cDNA synthesized from RNA obtained from mammary epithelial organoids (3 different animals per group) as discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036618#s4" target="_blank">Materials and Methods</a>. Lanes from left to right are: (lanes 1–3) 8 week virgin <i>Wwox WT</i>, (lanes 4–6) P18.5 <i>Wwox WT</i>, (lanes 7–9) 8 week virgin <i>Wwox KO</i>, and (lanes 10–12) P18.5 <i>Wwox KO</i>. <i>Wwox</i> expression is shown in the middle panel as further verification of <i>Wwox</i> ablation. <i>Gapdh</i> expression is shown as a normalization control. Each PCR reaction was performed at 24, 26, 28 and 32 cycles to ensure that reaction was in linear range. Results shown at 24 cycles.</p

<i>Wwox</i> mRNA expression in <i>BK5-Cre</i> model of Wwox deletion.

<p>qRT-PCR was used to determine the mRNA expression of <i>Wwox</i> in mammary epithelial organoids from <i>BK5 Wwox WT</i> and <i>KO</i> animals. The box plots show dramatically decreased <i>Wwox</i> mRNA levels in epithelium from 8 week virgin (A) and P18.5 days (B) <i>Wwox KO</i> mammary epithelium when compared to their respective <i>WT</i> counterparts. Mammary epithelial organoids were isolated from 3 different <i>WT</i> and 3 different <i>KO</i> mice in each group. Samples from each mouse were run individually in triplicate to assess <i>Wwox</i> expression. Samples were normalized to 18S expression.</p

Deletion of <i>Wwox</i> results in defective mammary branching and lobuloalveolar development.

<p>(A–B) Representative microphotographs obtained from whole mounts of 12 week virgin <i>BK5 Wwox WT</i> (A) and <i>KO</i> (B) mammary glands. The impaired branching phenotype was also dramatically evident in mammary gland from <i>MMTV KO</i> mice as illustrated in (C), extreme case of branching impairment in a 24 week virgin <i>MMTV Wwox KO</i> mammary gland. (D) The average number of branches/mm of ductal length was quantified for <i>BK5-Cre (−); Wwox^flox/flox</i> (Cre (−) WT, n = 4), <i>BK5-Cre (+); Wwox^+/+</i> (Cre (+) WT, n = 4), and <i>BK5-Cre (+); Wwox^flox/flox</i> (Wwox KO, n = 5). A small yet statistically insignificant (p = 0.06) decrease in branching can be seen in Cre(+) WT as compared to Cre(−) WT. The decreased branching found in <i>Wwox KO</i> mammary glands was significant when compared to either Cre (−) WT (p = 0.0002) or Cre (+) WT (p = 0.018). (E) Quantification of branches/mm ductal length in 24 week virgin <i>MMTV Wwox WT</i> controls (<i>MMTV-Cre (−); Wwox^flox/flox</i> and <i>MMTV-Cre (+); Wwox^+/+</i> were grouped together, n = 4) and <i>MMTV Wwox KO</i> (<i>MMTV-Cre(+); Wwox^flox/flox</i>, n = 4) mammary glands. Branching was statistically significantly decreased in <i>Wwox KO</i> when compared to <i>WT</i> controls (p = 0.017). Error bars indicate standard deviation.</p

<i>Wwox</i> deletion correlates with increased phospho-Stat3 protein in mammary epithelium.

<p>(A–B) Immunostaining for phospho-Stat3 (pStat3) protein in histological sections from 12 week virgin <i>BK5 Wwox WT</i> (A) and <i>Wwox KO</i> (B) mammary glands. Strong nuclear staining can be seen in the majority of cells in <i>Wwox KO</i> sections. (C) Quantification of percentage of cells positive for pStat3 staining in <i>Wwox WT</i> (n = 3) and <i>Wwox KO</i> (n = 3) histological sections. A total of 1000 cells were counted from random fields within each sample. (D) Whole cell lysates from mammary epithelial organoids from P18.5 <i>BK5 Wwox WT</i> (n = 3; WT4, WT5, WT6) and <i>Wwox KO</i> (n = 3; KO4, KO5, KO6) mice, were probed pStat3 levels. Wwox protein levels are shown to verify Wwox knockdown. Total Stat3 levels are shown as a control and Actin protein levels are shown as a loading control.</p

Conditional <i>KO</i> of <i>Wwox via BK5-Cre</i> and <i>MMTV-Cre</i>.

<p>(A–F) Immunostaining of formalin-fixed histological sections of mammary glands from each model of deletion reveals strong cytoplasmic Wwox staining in <i>WT</i> samples and significant loss of Wwox protein expression in <i>KO</i> animals. (A) 8 week virgin <i>BK5 Wwox WT</i>, (B) 8 week virgin <i>BK5 Wwox KO</i>, (C) P18.5 days <i>BK5 Wwox WT</i>, (D) P18.5 days <i>BK5 Wwox KO</i>, (E) 24 week virgin <i>MMTV Wwox WT</i>, (F) 24 week <i>MMTV Wwox KO</i>. Rabbit anti-Wwox <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036618#pone.0036618-Bednarek2" target="_blank">[14]</a> antibody was used at a 1∶50 dilution and counterstained with hematoxylin. All images were obtained at the same 10× magnification.</p