Fig 7. vIRF1 induces cellular transformation and angiogenesis by activating the SPAG9/JNK/VEGFA pathway.

(A). Soft agar assay of MM cells, KSHV-infected and transformed MM cells (KMM) and a mutant with a deletion of KSHV ORF-K9 infected MM cells (K9_Mut) (MOI of 3). The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. (B). CCK-8 assay of cells treated as in (A). (C). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in cells treated as in (A). (D). Soft agar assay of MM and KMM cells treated with the JNK inhibitor, SP600125 (50 μM) for 48 h. The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. (E). CCK-8 assay of cells treated as in (D). (F). Chicken chorioallantoic membranes (CAMs) assay of cells treated as in (D). (G). Matrigel plug assay in mice of cells treated as in (D). (H). Hematoxylin and eosin (H&E) staining analysis of histologic features (up; ×400) and immunohistochemical (IHC) staining analysis of the expression of SMA (down; ×400) in plugs in mice induced by cells treated as in (D). The newly formed blood vessels and the SMA expression were pointed out by black arrows, respectively. (I). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in HUVECs treated with PBS (PBS) or infected with wild-type KSHV (KSHV_WT) (MOI of 3) or vIRF1 mutant virus (K9_mut) (MOI of 3) followed by transduction with lentiviral vIRF1 at MOI 2 at 6 hpi. (J). CCK-8 assay of cells treated as in (I). (K). A schematic working model of the mechanism by which vIRF1 facilitates angiogenesis and cell transformation. vIRF1 enhanced SPAG9 transcription by interacting with Lef1 to promote the transcriptional activity of Lef1. Increased SPAG9 expression enhanced the activation of JNK1/2 pathway and VEGFA transcription contributing to KSHV-induced angiogenesis and tumorigenesis.