Figure 4

NRP1 promotes the invasion and metastatic dissemination of mCRPC cell models. (a) Flow cytometric quantification of NRP1 protein levels in benign (BPH-1, RWPE-1) and tumorigenic PCa cell lines. Error bars: s.d. n=3, *P <0.05. (b) Western blot of NRP1 expression in PC3-shCntrl, -shNRP1(1) and -shNRP1(2) total cell lysates. (c) Left panel: relative confluence of PC3-shCntrl (black lines), -shNRP1(1) (red lines) and -shNRP1(2) (blue lines) cells over 48 h measured by the CellPlayer Kinetic Proliferation assay. Right panel: DNA content in the same cell lines quantified by PicoGreen assay after 1, 3 and 5 days. (d) Representative phase-contrast images of PC3-shRNA models grown in 2D monolayer and (e) 3D On-top Matrigel cultures. Scale bars, 100 μm. (f) Quantification of vimentin and E-cadherin protein expression using the In-Cell Western technique (LI-COR) on intact PC3-shCntrl, -shNRP1(3) and -shNRP1(5) cells. Wells were stained immediately following wound scratch assays reported in Supplementary Figure 2. Bar chart displays combined intensity data (n=16 wells from three experiments). Error bars: s.d. (g) Vimentin and E-cadherin protein expression detected by immunofluorescence in PC3-shCntrl and -shNRP1(3) cells after 10 days of 3D On-top Matrigel culture. Red: actin; blue: DAPI. × 60 magnification. (h) Zebrafish-xenografted control (shCntrol) and NRP1 knockdown (shNRP1 (1) and shNRP1 (2)) PC3 cells (red fluorescent signal) at 1 day (left panels) and 5 days (right panels) post-injection (dpi). White arrows indicate metastatic dissemination outside of the yolk sac. (i) Percentage incidence of metastasis in xenografted zebrafish (n= 64, shContrl; n=30, shNRP1 (1); n=28, shNRP1 (2)). P=0.0002 (chi-square test).