Figure 3

PHA665752-induced activation of the mitochondrial apoptosis pathway. (A) Bax conformational change by PHA665752 treatment in PTC cell lines. B-CPAP and TPC-1 cells were treated with 5 [µmol/L PHA665752 for 4 and 8 h, respectively; cells were then lysed with 16 mmol/L CHAPS lysis buffer and immunoblotted with Bax rabbit polyclonal antibody. (B) Loss of mitochondrial membrane potential by PHA665752 treatment in PTC cells. B-CPAP and TPC-1 cells were treated with various doses of PHA665752 as indicated for 24 h. Live cells with intact mitochondrial membrane potential (□) and dead cells with lost mitochondrial potential (■) were measured by JC-1 staining and analyzed by flow cytometry as described in Materials and Methods. (C) PHA665752-induced cytochrome c release from mitochondria. B-CPAP and TPC-1 cells were treated with various doses of PHA665752 as indicated for 24 h. Mitochondria-free cytoplasmic fractions were isolated as described in Materials and Methods. Cell extracts were separated on SDS-PAGE, and immunoblotted with an antibody against cytochrome c and β-actin. (D) B-CPAP and TPC-1 cells were treated with various doses of PHA 665752 as indicated and cytoplasmic extracts were prepared, then 10 µg of protein from each sample was separated on SDS-PAGE, transferred to a PVDF membrane and immunoblotted with antibodies against XIAP and cIAP1. The blots were probed with an antibody against β-actin for equal loading.