Figure 3. AR activates PD-L1 transcription by binding to its promoter region. (A) Castration assay was performed in three HCC cell lines. (B) Predicted localization of AREs in PD-L1 promoter region (red). (C) Chromatin immunoprecipitation was performed in wild-type SK-Hep1 cells. The detecting primer was designed based on the prediction result of potential AREs. (D) Wild-type PD-L1 promoter construct was transfected into SK-Hep1 cells with internal control pRL-TK. Then, we performed luciferase reporter assays with manipulated AR to detect if AR could affect activation of PD-L1 promoter. (E) Luciferase reporter assays were performed after transfecting mutated 1st ARE into AR-overexpressed SK-Hep1 cells and AR knocked-down SK-Hep1 cells. *P<0.05, ** P<0.01 and *** P<0.001.