Upk3a^GCE/+ drives conditional expression of the human <i>AR</i> transgene in mouse bladder urothelium.

<p>(<b>A)</b> Schematic depicting the Cre/LoxP-mediated recombination, resulting in deletion of the STOP cassette from the <i>R26AR transgene</i> allele. (<b>B)</b> Experimental set-up for tamoxifen-induced human <i>AR</i> transgene expression. (<b>C)</b> Genomic PCR analyses of recombination events with specific primers for the human <i>AR</i> transgene allele (see Fig 2A) with bladder urothelium tissue samples of <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> and <i>R26AR</i>^{<i>LoxP/+</i>} mice. The AR trangene allele was detected in the samples of both <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> and <i>R26AR</i>^{<i>LoxP/+</i>} mice (black arrow), but TM induced recombination on the AR transgene allele was only observed in <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice (white arrow). (<b>D-F’)</b> Immunohistochemical staining of the bladder urothelium of TM injected <i>R26AR</i>^{<i>LoxP/+</i>} mice using either the antibody that is reactive to both mouse and human AR protein (m&hAR), <b>or</b> the antibody that is reactive to only human AR (hAR) (n = 9). <b>(F-G’)</b> Immunohistochemical staining of the bladder urothelium with the two different AR antibodies as described above using the samples of TM-injected <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice (n = 12). (<b>H-K”)</b> Single channel and merged immunofluorescent images of bladder tissues of TM-injected <i>R26AR</i>^{<i>LoxP/+</i>} and <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice stained with antibodies against CK5 p63, and the human AR (in red) (n = 6).</p

Conditional expression of the human <i>AR</i> transgene enhances BBN-induced carcinogenesis.

<p>(<b>A)</b> Experimental design to induce transgene expression and bladder carcinogenesis in <i>R26AR</i>^{<i>LoxP/+</i>} and <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i>. (<b>B-E)</b> Low (B-E) and high (B’-E’) magnification images of H&E staining depicting the range of pathological changes in <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice following TM injection and BBN treatment described in (<b>A)</b> (Hyperplasia:n = 30, Carcinoma in situ n = 5, Non-invasive carcinoma: n = 1, Invasive carcinoma: n = 17). (<b>F)</b> Analysis of the incidence of hyperplasia or urothelial cell carcinomas development in mice with different genotypes.</p

The effect of androgens on tumor development in <i>R26AR</i>^{<i>LoxP/+</i>} and <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i>mice.

<p>(<b>A)</b> Schematic depicting experimental design for castration of male mice and testosterone pellet insertion of female mice. (<b>B)</b> Analysis of the androgen effect on different genotypes of male and female mice with castration and testosterone pellet insertion. (<b>C)</b> Quantification of Ki67-positive cells in castrated and intact male mice and female mice with and without testosterone pellet (n = 4).</p

Upk3a^GCE/+ targets Cre recombination to bladder urothelium.

<p>(<b>A)</b> Schematic demonstrating Cre-mediated labeling of Upk3a^GCE/+-positive cells with the mT/mG reporter system. (<b>B)</b> Schedule of tamoxifen (TM) injection to induce Cre recombination of <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> mice. (<b>C-C’)</b> Lower and higher magnification images of labeled mT, mtd-Tomato, (red) or mG, mGFP, (green) in the bladder urothelium of <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> prior to TM injection (n = 4). (<b>D-D’)</b> Lower and higher magnification images of labeled mT (red) or mGFP (green) in the bladder urothelium in <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> following TM injection (n = 6). (<b>E-G)</b> Immunofluorescent images of bladder urothelium of TM injected <i>Upk3a</i>^<i>GCE/+</i><i>R26</i>^{<i>mTmG/+</i>} mice co-stained with antibodies against GFP (E-G”) with p63 (E’-E”), CK5 (F’-F”), or CK8 (G’-G”) (n = 6).</p