Analysis of the androgen receptor reporter construct in stably transfected cells.

<p>(<b>A</b>) Schematic representation of the ARE-tk-Luc androgen receptor reporter construct (hP = hPEST degradation signal). (<b>B</b>) Western blot analysis of steroid receptor expression in MCF-7, LNCaP and Du145 cells growing in full media. (<b>C</b>) Western blot analysis of PR expression in hormone starved MCF-7 cells -/+ E2 treatment for 24hr. (<b>D</b>), Luciferase activity from hormone treated LNCaP/Luc cells – treated for 24hours with 0-10nM mibolerone (Mib), estrogen (E2), progesterone (P4) and dexamethasone (Dex) (left hand side) and with 0-100nM of the androgens - (Mib), dihydrotestosterone (DHT), testosterone (Tes), androstenedione (A-dione) and dehydroepiandrosterone (DHEA) for 24 hours (right hand side). (<b>E</b>) Luciferase activity from hormone treated MCF-7/Luc cells – treated for 24hours with 0-10nM Mib, E2 or P4 or with additional 24hr pre-treatment with E2 (10nM) to induce PR expression. (<b>F</b>) Q-PCR quantification of relative expression of the steroid receptors AR, ER, and PR and luciferase transcripts in MCF-7/Luc cells, grown in full medium, transfected with siRNA against androgen receptor. (<b>F</b>) Luciferase activity from Du145/Luc cells treated with 0-100nM dex or mib (left hand side) or pre-transfected with an additional AR or empty vector expression constructs (right hand side). **P<0.01, *P<0.05 (t-test analysis).</p

Upper panel - Mouse tissues: A, prostate; B, testis; C, thymus; D, brain; E, skin and hair follicle; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, lacrimal gland; M, salivary gland; N, bone marrow; O, eye; P, heart.

<p>Lower panel - Human tissue: A, prostate; B, testis; C, thymus; D, brain; E, skin; F, kidney; G, liver; H, uterus; I, intestine; J, spleen; K, colon; L, breast; M, intestine and omentum; N, bone marrow; O, ovary and follicle; P, lung.</p

Androgen receptor reporter activity in transgenic mice.

<div><p>(<b>A</b>), Bioluminescent imaging of male and female ARE-Luc and C57/Bl/6J wild type mice, injected with 150mg/kg luciferin substrate, and imaged with a CCCD camera after 10 minutes. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm². </p> <p>(<b>B</b>), Luciferase enzymatic assays from tissue homogenates taken from ARE-Luc mice, normalised to protein content (Bradford assay). Male mice upper panel, female mice lower panel.</p></div

Analysis of AR and luciferase expression within a sample of mouse tissues.

<p>(<b>A</b>), Immunohistochemical co-localisation staining for AR (nuclear and cytoplasmic_ and luciferase (cytoplasmic) on consecutive formalin-fixed tissue sections taken from the ARE-Luc male mice. Upper panel represents image at 10x magnification, with box inset and lower panel showing 40x magnification. (B), Immunohistochemical staining for AR in a variety of female mouse tissues. </p

Bioluminescence analysis of <i>ex vivo</i> tissues.

<p><i>Ex vivo</i> bioluminescence imaging of tissues taken from ARE-Luc mice injected with 150mg/kg luciferin substrate and killed and dissected after 10 minutes. (<b>A</b>), Images of male tissues (upper panel) and female mice (lower panel). (<b>B</b>), Images of coronal sections through the cerebral cortex of a male and female mouse brain. CC - Cerebral Cortex, Th - Thalamus, Hth - Hypothalamus. (<b>c</b>), Image of the underside of the male brain, showing pituitary and optic nerve bundles. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence. Images are representative of the pattern seen in several mice.</p

Bicalutamide treatment reduces androgen receptor activity in male mice.

<p>Bioluminescent imaging of male mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. (A), Graph indicates measured bioluminescence signal from the gonadal region at the indicated timepoints; error bars represent the standard error from three mice in each group. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm². (C), panel showing <i>ex vivo</i> imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the testes and the prostate taken ex vivo, from male mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice. **P<0.01, *P<0.05 (t-test analysis).</p

Bicalutamide treatment reduces androgen receptor activity in female mice.

<p>(A), Bioluminescent imaging of female mice treated with bicalutamide (50mg/kg) for 24 and 48 hours. Graph indicates measured bioluminescence signal from the abdominal region at the indicated timepoints, error bars represent the standard error from three mice. (B), representative bioluminescent image of vehicle and bicalutamide treated mice at 48hours. Figure represents a greyscale photograph overlaid with a pseudocolour representation of bioluminescence; scale represents photons/sec/cm². (C), panel showing <i>ex vivo</i> imaging of the organs from bicalutamide treated mice after 48hours (intestines image not to scale). (D), Bioluminescence signal from the ovaries and the intestines taken ex vivo, from female mice treated for 48hours with vehicle or bicalutamide. Error bars represent the standard error from three mice, **P<0.01, *P<0.05 (t-test analysis).</p