<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>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

<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

Blood Chemistry Analysis.

<p>*Values are presented as the average±SEM.</p><p>**p-value using student's t-test comparing WT+HET vs. KO; NS-p>0.05.</p

Histomorphometric analysis.

<p><b>(A)</b> MicroCT images of femurs from postnatal day 18 mice. Note the dramatic difference in bone structure of the femur from KO mice vs WT and HET mice bones <b>(B)</b> Von Kossa stained sections of femurs from mice of the indicated genotypes. Black stained areas represent mineralized bone tissue. Note the significantly decreased mineralization in the KO mice bone sample <b>(C)</b> Quantitative histomorphometric analyses of postnatal day 18 day mice. We did not observe differences between wild-type and heterozygous mice, therefore, we compared KO data (n = 3) with WT and HET data combined (WT+HET, n = 5). Wild-type-WT, heterozygote-HET, knockout-KO. BV/TV%-bone volume/tissue volume, Md.V/TV%-mineralized volume/tissue volume, Tb.N-Trabecular number (1/mm), Tb.Th-Trabecular thickness (micrometers). *p<0.05.</p

<i>Wwox</i> KO mice have abnormal spleens and thymuses.

<p>Histopathology of spleens (top four panels) and thymuses (bottom four panels) from 18 day old KO and WT mice. Histological sections were stained with H&E. Note the significant atrophy of the spleen and the reduced cortical thickness in the thymus from the KO mice. Low power images of spleens (first row) and thymuses (third row) have a total magnification of 21X. High power images of spleens in (second row) have a total magnification of 84X and thymuses (bottom row) have a total magnification of 44X.</p

Genotypes of pups from <i>Wwox^+/ΔCre</i> × <i>Wwox^+/ΔCre</i> crosses.

<p>Genotypes of pups from <i>Wwox^+/ΔCre</i> × <i>Wwox^+/ΔCre</i> crosses.</p