Figure 3.
Effect of IGF-II treatment and WHV replication on HBx-mediated apoptosis. (A) IGF-II blocks HBx sensitization of cells to TNFα-mediated apoptosis. Chang cells transfected with HBx plasmids were treated with TNFα with or without 100 ng/ml IGF-II for 16 h starting 20 h posttransfection. Cells were fixed on coverslips, stained with Hoechst 33258, and photographed. (B) Colocalization of apoptosis to WHV replicating cells. Chang cells were transfected for 3 days with cDNAs encoding wild-type WHV or HBx mutant CWHV, treated with TNFα for 16 h, fixed and stained with Hoechst 33258 and anti-HBsAg fluorescein isothiocyanate antibodies, and then photographed (magnification ×600). HBsAg stained cells with CWHV(HBx−) (open arrows) and with wtWHV (HBx+) (filled arrows). 1(C) HBx expressed during in vitro WHV replication sensitizes cells to TNFα-induced killing. Chang cells were transfected for 3 days with a wild-type WHV pregenomic cDNA (pWHV) or an HBx(−) mutant (pCWHV) with or without 1.0 ng/ml TNFα and with or without 100 ng/ml human IGF-II for 20 h. Cell killing was quantitated by entry of trypan blue dye. Results present average of four independent experiments.