The non-recurrence rate of the IAD group and the CAD group with PSA ≤ 0.01 ng/ml.

<p>Kaplan-Meier analysis of progression-free survival in the patients who showed a PSA nadir of less than 0.01 ng/ml after LHRHa administration.</p

The median duration of time on treatment or time off treatment in the IAD group.

<p>The median duration of time on treatment or time off treatment in the IAD group.</p

Mean PSA level prior to posterior LHRHa therapy in the IAD group.

<p>Mean PSA level prior to posterior LHRHa therapy in the IAD group.</p

The non-recurrence rate and survival rate of the IAD group and the CAD group with Gleason Score ≤ 7.

<p>Kaplan-Meier analysis of progression-free survival (A) and overall survival (B) in both two groups in patients with Gleason Score ≤ 7.</p

Characteristics of patients treated with IAD and CAD.

<p>Characteristics of patients treated with IAD and CAD.</p

The non-recurrence rate and survival rate of the IAD group and the CAD group.

<p>Kaplan-Meier analysis of progression-free survival (A) and overall survival (B) in the IAD group and CAD group.</p

LY294002 inhibited TMV-promoted random motility and tube formation in NEC.

<p>(A) When treated by the PI3K inhibitor LY294002, cell motility was not stimulated by TMV (50 µg/ml) anymore. The results are presented as mean velocities ± SE (DMSO; n = 15, DMSO+TMV; n = 15, LY294002; n = 15, LY294002+TMV; n = 15). *<i>P</i><0.01. (B) In addition, LY294002 suppressed increase of tube formation in NEC treated by TMV (50 µg/ml). The results are presented as mean tube length per field ± SE. (DMSO; n = 10, DMSO+TMV; n = 10, LY294002; n = 10, LY294002+TMV; n = 10). *<i>P</i><0.01. These results indicate that TMV-induced proangiogenic phenotype is mediated by PI3K/Akt pathway, at least in part.</p

Characterization of isolated normal endothelial cells (NEC) from mice.

<p>(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.</p

TMV promote random motility and tube formation in NEC.

<p>Time-lapse observation revealed that TMV promote random motility in NEC. When treated by the endocytosis inhibitor dynasore, cell motility was not stimulated anymore. (A) Migrated trajectories of TMV-treated NEC (with/without 50 µM dynasore) were plotted and velocity was calculated using ImageJ. (B) The results are presented as mean velocities ± SE (DMSO; n = 15, DMSO+TMV; n = 15, dynasore; n = 15, dynasore+TMV; n = 15). *<i>P</i><0.01. TMV also promote tube formation in NEC. Dynasore canceled increase of tube formation even in NEC cultured with TMV. (C) Phase-contrast images of control and TMV-treated NEC (with/without 50 µM dynasore) cultured on a matrigel. Bar = 100 µm. Capillary-like structure enhanced with TMV. Total length of capillary-like tubes was analyzed using ImageJ. (D) The results are presented as mean tube length per field ± SE. (DMSO; n = 10, TMV; n = 10, dynasore; n = 10, dynasore+TMV; n = 10). *<i>P</i><0.01.</p

TMV activate the PI3K/Akt pathway.

<p>(A) TMV activated Akt in NEC. NEC were incubated with TMV (50 µg/ml) for the indicated time periods, and phosphorylated-Akt (p-Akt) and total-Akt (t-Akt) levels were determined in cell lysates by immunoblotting. Lower; Relative phosphorylated-Akt levels (p-Akt/t-Akt). (B) LY294002 inhibited Akt phosphorylation induced by TMV. NEC were preincubated with or without LY294002 (20 µM) for 2 h, then with or without TMV (50 µg/ml) for 20 min. Relative phosphorylated-Akt levels were quantified by immunoblotting. (C) The endocytosis inhibitor blocked Akt phosphorylation induced by TMV. NEC were preincubated with or without dynasore (20, 40 µM) for 30 min, and then with or without TMV (50 µg/ml) for 20 min. Relative phosphorylated-Akt levels were quantified by immunoblotting.</p