Figure 9

Manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-augmented apoptosis is involved in the activation of caspases. (A) SKOV-3 cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with the extracellular signal-regulated kinase (ERK)1/2 inhibitor (PD98059, 50 μ M) for 30 min followed by doxorubicin (DOX, 5 μ M) or paclitaxel (PTX, 0.1 μ M) addition for another 48 h. Cells were then analysed for expression of caspase-9, caspase-3, and caspase-8 proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control. (B) Cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with the pan-caspase inhibitor (Z-VAD-FMK, 50 μ M), caspase-8 inhibitor (Z-IETD-FMK, 10 μ M), or caspase-9 inhibitor (Z-LEHD-FMK, 50 μ M) for 3 h, followed by DOX or PTX addition for another 48 h. Cells were then harvested for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Experiments were repeated three times, and data are shown as mean±s.d. ^*P<0.05 vs untreated control cells. (C) Cells were transiently transfected with the control vector (pcDNA3.1) or FLAG-tagged dominant-negative mutant of caspase-9 (DN-Casp9) for 24 h, and then treated with DOX or PTX for another 48 h. Whole-cell lysates were analysed for expression of DN-Casp9 by western blotting using FLAG-specific antibodies. β-Actin serves as a protein-loading control. Right, apoptosis was assessed by the TUNEL assay. Experiments were repeated three times, and data are shown as mean±s.d. ^*P<0.05 vs untreated MnSOD siRNA-transfected cells as indicated.