Prostate Cancer Angioprevention by Flavonolignans Figure 3. Flavonolignans decrease VEGFR1, HIF-1a, phosphorylated and total Akt levels in DU145 xenografts. DU145 xenograft tissues were analyzed for VEGFR1, HIF-1a, phosphorylated Aktser473 and total Akt levels by IHC. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. The data shown in the bar diagrams is the mean 6 SEM of 4–5 samples. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p # 0.001. doi:10.1371/journal.pone.0034630.g003 Ex Vivo Capillary Formation Assay Aortas isolated from mice were cleaned, cut into small fragments and placed on matrigel-covered wells and covered with another 100 mL matrigel. After these aortas were cultured for 24 h, the medium (complete HUVEC media) was replaced with or without flavonolignans (30 and 60 mM). Treatments were replaced after 48 h. After 6 days of total incubation, vessels sprouting from the aortas were photographed using Cannon Power Shot A640 camera on Zeiss inverted microscope. Tube Formation Assay HUVEC (4 6104) were cultured in 1 mL EBM2 (supplemented with 0.5% FBS and 4 ng/mL VEGF) with various diastereoiso- mers (5–30 mM) on matrigel coated plates. After 9 h of incubation, tubular structure formation was quantified by calculating the tube length (at 100x) with Zeiss inverted microscope using Cannon Power Shot A640 camera and AxioVision Rel.4.7 software. In a related experiment, DU145 cells were treated with 30 mM dose of each flavonolignan for 72 h, and fresh media (0.5% FBS) was added and collected after 12 h (labeled as ‘conditioned media’). The conditioned media mixed with 0.5% FBS supplemented EBM2 media (75:25 ratio) was then added to HUVEC and tube formation was studied as described above. Cell Viability Assay HUVEC were treated with or without VEGF (10 ng/mL) and different concentrations of flavonolignans (5–30 mM). After the desired treatment, total cell number was determined using a hemocytometer. Transwell Invasion Assay In this assay, the bottom chambers of Transwell were filled with EBM2 media containing 0.5% FBS supplemented with 4 ng/mL VEGF, and in the top chambers HUVEC (4 6104) were seeded in 500 mL EBM2 (0.5% FBS) plus 30 mM dose of each flavonolignan. After 10 h, invasive cells were quantified as described previously [30,31]. Similar assay was also performed with DU145 cells plated in the bottom chamber (RPMI media with 0.5% serum) and HUVEC (EBM2 media with 0.5% serum) in the upper chamber. In two separate experiments, flavonolignans were added either in the upper chambers or in the lower chambers and invasiveness of HUVEC was studied in each case. Cell Cycle Distribution and Apoptosis Cell cycle distribution (saponin/PI staining) and apoptosis (Annexin-PI staining) were analyzed by FACS [32,33]. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34630