Figure 3

Conditioned media from B4rv- or XMRV-infected LNCaP cells induce an immature SMC phenotype and promotes HUVEC tube formation in vitro . A. QQ-rtPCR for expression of SMC marker genes Tagln, Myh11, and matrix metalloproteinase genes Mmp3 and Mmp9, in rat aortic SMCs after culture in tumour conditioned media (TCM) of uninfected LNCaPs, or 4070a-MoMLV-, XMRV-, and B4rv-infected LNCaP cells, demonstrating that SMCs down-regulate the expression of SMC marker genes when cultured in the TCM of LNCaPs infected with XMRV or B4rv but not those infected with MoMLV-4070a. TCM was generated by first applying viral particles of equivalent titers onto uninfected LNCaP cells for 24 hours, then culturing LNCaPs in serum-free media for 3 days. Media was collected and filtered through a 0.45μm pore to remove cellular debris, and then applied onto SMCs (see Methods). B. Migration of rat aortic SMCs across a transwell membrane in the presence of the TCM from uninfected LNCaPs, or 4070a-MoMLV-, XMRV-, and B4rv- infected LNCaP cells, showing that SMCs cultured in the TCM of LNCaPs infected by XMRV or B4rv exhibit increased migration as compared to SMC treated with TCM from uninfected LNCaPs. SMC culture media without serum (SFM) was used as a negative control, and culture media with 10% serum was used as a positive control. C. Tube formation assay of HUVECs cultured in Matrigel in the presence of the TCM from uninfected LNCaPs, and XMRV- or B4rv- infected LNCaP cells, demonstrating that HUVECs cultured in the presence of TCM from XMRV- or B4rv-infected LNCaPs form more tubes, based on number of branch points per field, than those cultured in the TCM of uninfected LNCaPs. HUVECs were treated with either siVEGF or a non-target siRNA control before plating in TCM and Matrigel, demonstrating that XMRV-infected LNCaP TCM induced HUVEC tube formation is VEGF-dependent.