Figure 2. Transcription factor YY1 is required for HBV-activated Sox4 expression.

(A) Sox4 promoter activity was detected in three cell lines co-transfected with pBlue-HBV1.3 or pBlue and pSox4-Luc. Luciferase activity was measured using a TD-20/20 luminometer and normalized to the control. The results are presented as means ± SD (n = 3). (B) The potential YY1 binding sites on Sox4 promoter were shown (upper panel). ChIP assays demonstrate that YY1 binds to the three potential binding sites on Sox4 promoter (lower panel). (C) Sox4 promoter containing three YY1 binding sites and its mutant promoters carrying deleted YY1 sites were generated and sub-cloned into pGL3-Basic to create pSox4-Luc and its mutant pSox4-Luc (M1-M5) (left panel). Cells were co-transfected with the indicated plasmids. Luciferase assays were performed and normalized to the control (right panel). The results were presented as means ±SD (n = 3). (D) HepG2 cells were co-transfected with indicated plasmids and siRNA mimics. Luciferase activity was measured and normalized to the control. The results were presented as means ±SD (n = 3). (E) HepG2.2.15 cells were transfected with siR-NC or siR-YY1 mimics, while HepG2 and Hep3B cells were co-transfected with pBlue or pBlue-HBV1.3 and siR-YY1 or siR-NC. The indicated proteins were analyzed by Western blotting. (F and G) YY1 mRNA (upper panel) and protein (lower panel) levels were detected in HepG2.2.15 compared with HepG2 cells or in HBV-transfected cells compared with pBlue-transfected cells. *P < 0.05, **P < 0.01.