Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway
Fig 2
vIRF1 transcriptionally activates SPAG9 by interacting with Lef1.
(A). RT-qPCR analysis of mRNA level of SPAG9 in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (B). RT-qPCR analysis of mRNA level of SPAG9 in treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV) (MOI of 3). (C). Luciferase reporter assay of the activity of SPAG9 promoter in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (D). Luciferase reporter assay of the activity of SPAG9 promoter in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). (E). Putative binding sites of Lef1 in the promoter region of SPAG9 gene. (F). ChIP assays of SPAG9 promoter. Immunoprecipitation was performed in vIRF1- or pHAGE-transduced HUVECs with anti-Lef1 antibody. Both SPAG9 primers (1) and (2) were used to amplify the sequences of the above two putative binding sites of Lef1 in the region of SPAG9 promoter as described in (E). (G) and (H). ChIP assays of SPAG9 promoter. Immunoprecipitation was performed in vIRF1-transduced HUVECs with anti-Flag antibody. Isotype IgG, anti-RNA polymerase II antibody and amplification of GAPDH promoter were used to examined the work of system. (I). RT-qPCR analysis of mRNA expression level of SPAG9 in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). (J). Western-blotting analysis of SPAG9 expression in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). Data were shown as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t-test.