Figure 4

Effect of PHA665752 on TRAIL-induced apoptosis in PTC cells. (A) PHA665752 induces upregulation of DR5. TPC-1 cells were treated with 10 µmol/L PHA665752 for the indicated period, then cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane and immunoblotted with antibodies against DR5. The blots were probed with an antibody against β-actin for equal loading. (B) Effect of NAC on PHA665752-induced DR5 upregulation in PTC cells. B-CPAP and TPC-1 cells were pretreated with 10 mmol/L NAC for 2 h and subsequently treated with 5 µmol/L PHA665752 for 24 h. RT-PCR: total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcripted with random hexamers. RT-PCR and relative quantification real-time RT-PCR were performed as described in Materials and Methods. Western blot: after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to an immobilon membrane and immunoblotted with antibodies against DR5 and β-actin. *P < 0.01 (PHA665752 treatment versus control); **P < 0.001 (NAC + PHA665752 treatment versus PHA665752 treatment alone). (C) B-CPAP cells were transfected with or without scrambled siRNA (100 nmol/L) and DR5 siRNA (100 nmol/L) for 48 h. After 48 h, cells were treated with or without indicated doses of PHA665752 for 24 h, and then apoptosis status was detected by flow cytometer using Annexin/PI staining. Columns, mean of three independent experiments; bars, SD. (D) B-CPAP cells were transfected with or without 100 nmol/L scrambled siRNA and 100 nmol/L DR5 siRNA for 48 h. After 2 d, cells were treated with or without indicated doses of PHA665752 for 24 h, then cells were lysed and proteins were immunoblotted with antibodies against DR5, caspase-3, PARP and β-actin. (E) B-CPAP and TPC-1 cells were treated with 2.5 µmol/L PHA665752 alone, 2.5 ng/mL TRAIL alone and two agents in combination for 24 h, and cells were subsequently stained with fluorescein-conjugated Annexin V and propidium iodide and analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SD. *P < 0.001. (F) B-CPAP and TPC-1 cells were treated with 2.5 µmol/L PHA665752, 2.5 ng/mL TRAIL and a combination of 2.5 µmol/L PHA665752 with 5 ng/mL TRAIL for 24 h. Then cells were lysed and equal amounts of proteins were separated on SDS-PAGE, transferred to a PVDF membrane and immunoblotted with antibodies against p-Met, caspase 3, cleaved caspase 3, Bcl-XL, PARP and β-actin.