AdRspo1 treatment induces β-catenin activation in irradiated crypts.

<p>Representative immunoblot (<b>Fig. 7A</b>) and densitometric analysis (<b>Fig. 7B</b>) of nuclear/cytosolic ratios of β-catenin from AdRspo1 and AdLacZ treated cohorts after WBI(10.4Gy). Nuclear fraction purity was validated by the absence of β-tubulin, while the purity of the cytosolic fraction was evaluated by the absence of PCNA (Fig. 7A). A continuous decline in nucear/cytosolic ratios of β-catenin was predominate in samples from irradiated AdLacZ cohorts. This is further supported by the densitometric analysis of β-catenin expression (Fig. 7B) from the nuclear/cytosolic ratio demonstrating the significant differences in AdRspo1 when compared to AdLacZ treated mice prior to (Day –1) until Day +5 post WBI.</p

Xylose absorption assay.

<p>A time course study (1–10dys) showed significant recovery (p<0.002) of xylose absorption at 3.5 to 7 days in AdRspo1-treated cohorts, when compared to AdLacZ controls, thereby indicating the functional regeneration of intestine after radiation injury. AdLacZ-treated animals were incapable of demonstrating adequate xylose absorption after radiation injury, further contributing to animal mortality.</p

AdRspo1 treatment has no effect on tumor growth.

<p>Effect of AdRspo1 and AdLacZ treatment on tumor growth rate of Balb/c mice (n = 5) irradiated with 14Gy ABI. Significant delay in tumor growth (p<0.0001) was observed in ABI groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008014#pone-0008014-g003" target="_blank">Fig A</a>) compared to untreated mice.</p

AdRspo1 increases the number of regenerative crypts in irradiated mice.

<p>Effect of AdRspo1 and AdLacZ treatment on intestinal crypt depth (A), proliferation rate (B), apoptotic cells (C) at 1day and 3.5 days after WBI and the number of regenerative crypts (D) at 3.5 days after WBI. A representative sampling of thirty crypts was assessed for each treatment group.</p

Histolological assessment of intestine after Irradiation.

<p>H&E staining demonstrates increased crypt depth and increased villi thickness in AdRspo1-treated animals following exposure to WBI. BrdU immunohistochemistry demonstrates higher crypt cell proliferation after AdRspo1 treatment when compared to AdLacZ cohorts. Finally, TUNEL staining demonstrates a decrease in the rate of TUNEL-positive, apoptotic cells in AdRspo1-treated mice post-WBI, when compared to intestinal lumen of AdLacZ-treated mice.</p

AdRspo1 treatment increases the number of Lgr5-positive intestinal stem cells in irradiated crypts.

<p>Immunohistochemical staining of Lgr5 in murine jejunum crypts at 3.5 days prior and after WBI. There was an increase in the number of Lgr5 postive cells at crypt columnar base in AdRspo1 treated cohorts when compared to AdLacZ (magnification 60x; <i>arrows</i>).</p

Time course evaluation of serum Rspo1 expression.

<p>(<b>A</b>) Treatment schema: AdRspo1 or AdLacZ (5×10⁹ pu) was injected intravenously 3 and 1 day before WBI (10.4 Gy) in C57Bl/6 mice. Animals were followed for survival and histological endpoints. (<b>B</b>) Immunoblots of murine serum demonstrating time course evaluation of serum Rspo1 expression after WBI. (<b>C</b>) Representative immunoblot of serum Rspo1 levels in C57Bl/6 mice, following treatment with AdRspo1 + WBI.</p

AdRspo1 treatment protected C57Bl/6 mice from radiation-induced mortality.

<p>Kaplan-Meier survival of C57Bl/6 mice treated with AdRspo1 or AdLacZ prior to WBI (10.4Gy) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008014#pone-0008014-g002" target="_blank">Fig 2A</a>) and 12–16 Gy AIR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008014#pone-0008014-g002" target="_blank">Fig 2B</a>) . Note a significant (p<0.003) increase in median survival in AdRspo1-treated mice with a median survival time of 27±1.6 days, compared to AdLacZ cohorts, 10±1.4 days. With 12–14Gy AIR median survival time for AdLacZ treated animals is 13±1.2 and 11±1.6 days compared to 25±1.3 and 19±1.4 in AdRspo1-treated animals.</p

Regiospecific liver repopulation in DPPIV⁻ recipients by donor DPPIV⁺ hepatocytes following conformal HIR of one half of the median lobe.

<p>All liver lobes, except one half of the median lobe were shielded from HIR. Three months after hepatocyte transplantation, liver sections were stained histochemically for DPPIV activity. A region at the junction of the irradiated and non-irradiated regions of the median lobe is shown. Panel a. Low power view showing clusters of DPPIV-positive donor hepatocytes (red, white arrow) in the upper part of the section that had received preparative HIR, and the absence of such clusters in the lower part of the section that had been shielded from HIR. Panel b. High power view showing a donor hepatocyte cluster. The black bars indicate 100 µm.</p

Ugt1a1 activity toward bilirubin in Gunn rats that received conformal HIR before hepatocyte transplantation.

<p>Ugt1a1 activities in liver homogenates of different recipient groups as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046775#pone-0046775-g002" target="_blank">Figure 2</a> are shown (means±SD) as percent of the activity in the liver of Wistar-RHA rats (2.6 µmol/g liver per hour).</p