Differential responses to carboplatin treatment in PC9 and A549 cells.

<p>A: Carboplatin-induced growth inhibition in PC9 and A549 cells using a sulforhodamine B assay 72 h post treatment. B: Western blot analysis of the levels of uncleaved PARP, cleaved PARP and cleaved (active) caspase 3 72 h post carboplatin treatment (0–200 µM) in PC9 and A549 cells. Actin was used as a loading control. C, D: Flow cytometric analysis of PC9 (C) and A549 cells (D) treated with carboplatin (100 µM) or vehicle. Apoptotic cells were identified by Annexin V-Alexafluor488 (λ Ex/Em = 495/519 nm) and necrotic cells by 7-AAD (λ Ex/Em = 546/647 nm). Population Q4 represents viable cells, whereas population Q3 represents apoptotic cells that have low 7-AAD fluorescence and stain with Annexin V. Population Q2 represents secondary apoptotic/necrotic cells.</p

Temporal changes in cell death markers and ¹⁸F-ICMT-11 uptake after carboplatin treatment.

<p>A: Time course of changes in caspase 3/7 activity following carboplatin treatment. B: Western blot analysis of the levels of uncleaved PARP, cleaved PARP and cleaved (active) caspase 3 post 50 µM carboplatin treatment (0–96 h) in PC9 (i) and A549 cells (ii). C: Temporal changes in ¹⁸F-ICMT-11 uptake in cells following carboplatin treatment. D: Correlation between caspase 3 activity and ¹⁸F-ICMT-11 uptake in PC9 cells.</p

¹⁸F-ICMT-11 cell uptake correlates to dose-dependent increases in caspase 3 activity following carboplatin treatment.

<p>A: Chemical structure of ¹⁸F-ICMT-11. B: Dose-dependent changes in caspase 3/7 activity following carboplatin treatment. C: Dose-dependent changes in ¹⁸F-ICMT-11 uptake in cells following carboplatin treatment. D: Correlation between caspase 3 activity and ¹⁸F-ICMT-11 uptake in PC9 cells.</p

Voxel-wise analysis of ¹⁸F-ICMT-11 PET imaging data by PVIS.

<p>The intensities of all voxels within the tumour ROIs were computed and expressed as histogram plots of normalized voxel intensity versus the number of voxels. A, B: Typical data from three representative animals (vehicle, 24 h or 48 h carboplatin-treated) for PC9 (A) and A549 (B) are shown. C, D: The statistical comparison of 95^th percentile voxel intensities for PC9 (C) and A549 (D) was performed using Prism v5.0 software (GraphPad). Mean ± SD (<i>n</i> = 4–6 animals per group).*, <i>P</i><0.05; **, <i>P</i><0.01.</p

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

Experimental design of the ERα-AIB1 split luciferase assay.

<p>Estrogen (E2) binding promotes ERα-AIB1 interaction and consequent reconstitution of the N- (NLuc) and C-terminal (CLuc) portions of the split firefly luciferase. In the case of anti-estrogens (AE), the interaction may be dependent on the tissue and AE context.</p

Tumour active caspase-3 and TUNEL immunohistochemistry analysis.

<p>Tumour tissues were removed after PET imaging scan, processed for histological analysis and stained for active (cleaved) caspase-3 and DNA fragmentation (TUNEL assay) detection, in conjunction with H&E staining. A: Representative images of histological tumour sections are shown. Staining intensities for cleaved caspase 3 (B) and TUNEL (C) were determined using the ImageJ software and expressed as percent staining per field. Data are mean ± SD. *, <i>P</i><0.05; ***, <i>P</i><0.001. <i>n</i> = 3 tumour sections with 5 random FOV per section. Photographic images of H&E-stained sections were acquired at 100×, with all other images acquired at 400×. Scale bar = 200 µm. Abbreviations: N, necrotic; V, viable.</p

¹⁸F-ICMT-11 PET image analysis of PC9 and A549 xenografts in vehicle and carboplatin-treated mice.

<p>A, B: Tumour volumes recorded by calliper measurements of PC9 (A) and A549 tumours (B) pre- and post-carboplatin treatment as indicated. Data shown are mean ± SD of % volume compared to baseline (<i>n</i> = 4). *, <i>P</i><0.05; **, <i>P</i><0.01. C, D: Representative axial PET-CT images (30–60 min summed activity) for PC9 (C) and A549 (D) tumours. Tumour margins, indicated from CT image, are outlined in red. Mean ± SD (<i>n</i> = 4–6 animals per group). E, F: The tumour TAC representing average counts from a dynamic 60-minute scan for PC9 (E) and A549 xenografts (F) following carboplatin treatment (vehicle, 24 h or 48 h carboplatin-treated; <i>n = </i>4–6 animals per group).</p

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

Time-course of E2 induced ERα-AIB1 luciferase fragment complementation.

<p>293 cells transiently transfected with N-S-AR/EL-S-C (A) or N-EL/AR-S-C (B) were treated with vehicle or 1 μM E2 for 2–48 hours prior to quantification of luciferase activities. Inset graphs indicate change in luciferase activity after 15 minutes E2 incubation. Graphs show the ratio of firefly luciferase relative to <i>Renilla</i> luciferase activity (± SEM triplicates). ANOVA was used to determine statistical significance relative to vehicle (*** p≤0.001, ** p≤0.01).</p