Fig. 4. vIRF1 transcriptionally activates CDCP1 by upregulating Lef1 and binding to Lef1.

a qPCR analysis of CDCP1 mRNA expression in HUVECs transduced with 2 MOI lentiviral vIRF1 (vIRF1) or control lentivirus (pHAGE). b qPCR analysis of CDCP1 mRNA expression in HUVECs treated with PBS (PBS) or infected with wild-type KSHV (KSHV_WT). c Luciferase reporter assay of the activity of CDCP1 promoter in HUVECs transduced with 2 MOI lentiviral vIRF1 (vIRF1) or control lentivirus (pHAGE). d Western blotting analysis of Lef1 and CDCP1 expression in HUVECs transduced with 2 MOI lentiviral vIRF1 (vIRF1) or control lentivirus (pHAGE). e Immunohistochemical staining of Lef1 in normal skin, skin KS of patient #1 (Skin KS1), skin KS of patient #2 (Skin KS2), and skin KS of patient #3 (Skin KS3). Magnification, ×200, ×400. f Results were quantified in e. g Western blotting analysis of CDCP1 expression in lentiviral vIRF1- or control pHAGE-infected HUVECs, followed by transduction with lentivirus expressing a mixture of shRNAs targeting Lef1 (shLef1). h qPCR analysis of CDCP1 mRNA expression in lentiviral vIRF1- or control pHAGE-infected HUVECs, followed by transduction with lentivirus expressing a mixture of shRNAs targeting Lef1 (shLef1). i Luciferase reporter assay of the effect of Lef1 on transcriptional activity of CDCP1 promoter. j Luciferase reporter assay of the activity of CDCP1 promoter in lentiviral vIRF1- or control pHAGE-infected HUVECs, followed by transduction with lentivirus expressing a mixture of shRNAs targeting Lef1 (shLef1). k ChIP assays of CDCP1 promoter. Immunoprecipitation was done in HUVECs transduced with 2 MOI lentiviral vIRF1 (vIRF1) or control lentivirus (pHAGE) with anti-Lef1 or anti-Flag antibodies. l, m Immunoprecipitation analyses of the interaction between vIRF1 and Lef1. The light chain band was used as a control to show the equal amounts of antibodies used for immunoprecipitations. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test.