FIG. 6.
XIAP antagonists induce caspase 9 activation and apoptosis in Apaf-1−/− MEFs. (A) pSmacΔMTS-IRES2-eGFP (pSmac) induces apoptosis in STS-treated Apaf-1−/− MEFs. The indicated cultures were first transfected with pSmac or empty vector (pGFP) and were then treated with STS alone or STS plus embelin for 4 h. GFP-positive cells were gated and presented as forward scatter (FSC) versus annexin V-PE. (B) Primary MEFs were treated with 2 μM STS and the indicated concentrations of embelin for 4 h; caspase 9 enzymatic activity was assessed using the fluorogenic substrate LEHD. RFU, relative fluorescence units. (C) The indicated MEFs were treated with vehicle or 30 μM embelin for 4 h and then assessed by an annexin V binding assay. (D) Embelin induces apoptosis in STS-treated Apaf-1−/− MEFs. The indicated MEF and MEM cultures were treated for 4 h with vehicle (dimethyl sulfoxide) alone, 2 μM STS, or 2 μM STS plus 30 μM embelin (STS/Emb). Apoptotic response was assessed by an annexin V assay. (E) Embelin cooperates with E2F1 to induce apoptosis in Apaf-1−/− MEFs. Apaf-1−/− MEFs were infected with Ad.E2F1-IRES-eGFP. The cultures were treated with vehicle alone or vehicle plus embelin (30 μM) and stained 24 h later with annexin V-PE. Relative apoptosis (annexin V-positive cells) normalized for infection efficiency (GFP-positive cells) is indicated by the AnnexinV/GFP ratio. ctrl., control. (F) Embelin induces caspase 9 enzymatic activity in STS-treated Apaf-1−/− MEFs. Wild-type or Apaf-1−/− MEFs were treated with vehicle alone, STS, or STS plus embelin for 4 h. Bar graph and standard error bars represent the corrected means of six independent experiments and the corresponding standard deviations. (G) Embelin induces caspase 9 processing in STS-treated Apaf-1−/− MEFs. Wild-type or Apaf-1−/− MEFs were treated with vehicle alone, STS, or STS plus embelin for the indicated intervals and then analyzed by Western blotting with caspase 9-specific antibodies. Emb., embelin; casp., caspase.