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IJC_24778_sm_suppfig1.tif15603KFigure S1. AAV-ARHP8 suppressed tumor growth in C4-2 xenografts. C4-2 cells (2 × 106 cell per injection) were used to establish xenografts in castrated nude mice. Once the xenografts were palpable (30-50 mm3), AAV-ARHP8 or AAV-GFP viruses were intra-tumorally injected at a dose of 1.0 × 109 viral particles per 50 mm3 tumor volume (day 0). (A) Tumor growth was monitored for 8 weeks. (B) Analysis of GFP distribution, AR expression, BrdU incorporation and TUNEL labeling. Frozen sections were used for assessing GFP distribution under fluorescent microscope. Immunostaining for AR and BrdU and TUNEL assay were carried out on paraffin sections as described earlier. Magnification of image, × 200. (C) Quantitative data (Mean ± SEM) were presented from panel B. The asterisk indicates a significant difference compared to the control (ANOVA analysis for panel A, P < 0.05; Student t-test for panel C, P < 0.01, n = 8).
IJC_24778_sm_suppfig2.tif3132KFigure S2. Tissue and cellular distribution of AAV-ARHP8 virus. (A) 22Rv1 xenograft tumors were established in 3 nude mice. Once tumors were palpable (30 mm3), animals were received viral injection of 2.0 × 1011 AAV-ARHP8 viral particles via tail vein. Three days later, xenograft issue or organ samples were harvested from animals. Total RNAs were extracted using TriZolTM reagent, and equal amount of RNA samples from the same organ or tissue types of individual animals were pooled together for the next experiments. RT-PCR was performed using RETROscritpTM kit to determine GFP expression. S28 gene expression was used as internal control. (B) Ultra-structural evaluation of viral distribution in xenograft tumors by transmission electronic microscopy (TEM). After dissection, 22Rv1 xenograft tumors from systemic treatment with AAV-ARHP8 or AAV-GFP viruses (obtained from the experiment shown in Fig 4D) were chopped into small pieces (3 × 3 mm), fixed and processed for TEM evaluation. Viral particles were readily visible as pointed by black arrows (AAV) based on the morphologic feature of the viral vesicles as described.31 Please note also that in AAV-ARHP8-treated tumor cells (panel c & d), typical apoptotic features including nuclear shrinkage and membrane blebbing are obviously visible. In contract, xenograft tumors after AAV-GFP treatment (panel a & b) show normal morphology as evidenced by smooth membrane and even layout of nuclear contents. AAV, adeno-associated virus; Mit, mitochondria, Nu, nuclear compartment.

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