HBZ activates TGF-β signaling dependent on p300. (A) E1A repressed HBZ-induced activation of TGF-β. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pME18Sneo-sHBZ (20 ng), and pCS2+-E1A or pCS2+-E1A-ΔNT (2 ng). Luciferase activity was measured 24 hours after stimulation by TGF-β (0, 10 ng/mL). (B) HBZ synergized with Smad3 and p300 to enhance TGF-β. HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pcDNA3.1-mycHis-sHBZ (200 ng), FLAG-Smad3 (50 ng), and pCMV-p300 (2, 5 μg). At 24 hours after transfection, the cells were treated with or without TGF-β (10 ng/mL). Luciferase activity was measured after 24 hours. Expression of sHBZ, Smad3, and p300 was detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel). (C) HBZ, Smad3, and p300 could form a ternary complex. mycHis-sHBZ (4 μg), FLAG-Smad3 (4 μg), and HA-p300 (4 μg) were cotransfected into COS7 cells, which were subsequently treated with TGF-β (5 ng/mL). Ternary complexes were detected by sequential immunoprecipitation with anti-FLAG agarose affinity gel and anti-HA antibody, followed by immunoblotting with the His antibody. (D) HBZ enhanced the interaction between Smad3 and p300. COS7 cells were cotransfected with mycHis-sHBZ (4 μg), FLAG-Smad3 (4 μg), and HA-p300 (4 μg). Cell lysates (samples from the experiment of Figure 4E) were subjected to immunoprecipitation using anti-HA followed by immunoblotting with anti-FLAG. (E) sHBZ, Smad3, and p300 bind to the Smad-responsive promoter. After transfection with mycHis-sHBZ, FLAG-Smad3, and HA-p300, and treatment with 5 ng/mL of TGF-β for 24 hours, HepG2 cells were chromatin immunoprecipitated by each indicated antibody. The precipitated DNAs and 1% of the input cell lysates were amplified by the 3TP promoter specific primers. Expression of sHBZ, Smad3, and p300 was detected by Western blot (bottom panel).