Fig. 8: TIP60 and ΔNp63α inhibit cell death and apoptosis in cisplatin-resistant cells.

A A431 Pt cells (left-panel) transfected with NSC, sip63 and siTIP60 and harvested at 48 h post transfection. A431 Pt cells (right-panel) were treated with either Vehicle (DMSO) or 50 μM of TH1834 as indicated. Cells were harvested, total RNA extracted, and PUMA transcript levels were measured by TaqMan-based qRT-PCR. The y-axis indicates the fold change in transcript levels relative to the corresponding NSC or vehicle (Veh) control. Error bars indicate means + 1SEM from three independent experiments. B A431 Parental and Pt cells were transfected with NSC, sip63 or siTIP60 and analyzed by flow cytometry at 48 h post transfection. The y-axis indicates the percentage of cells in Sub-G1 cell-cycle phase. Error bars indicate the mean + 1SEM from three independent experiments. Immunoblot analysis (bottom panel) was performed to measure the changes in ΔNp63α and TIP60 protein levels and confirm silencing. Immunoblot analysis was performed using antibodies specific for p63, TIP60 or β-actin. β-actin was included as a loading control for equivalent protein. Representative blot is shown. C A431 Parental and Pt cells were transfected with NSC, sip63 and siTIP60. Cells were pulsed with vehicle or 42 μg/ml of cisplatin for 2-h and were subjected to immunoblot analysis 48 h later. D A431 Parental and Pt cells pulse treated with vehicle or 42 μg/ml of cisplatin for 2-h followed by continuous treatment with either vehicle or TH1834 (50 μM) for 24 h. Immunoblot was performed using antibodies specific for γ-H2AX, PARP, cleaved PARP, cleaved caspase-3, p63, TIP60 or β-actin. Fold change in ΔNp63α protein relative to respective NSC or vehicle-treated control is listed above each band in (B, C, D). β-actin was included as a loading control for equivalent protein in each lane. Densitometric analysis (top panels) showing the fold change in cleaved-PARP and γ-H2AX levels normalized to β-actin. Error bars indicate as mean + 1SEM from three independent experiments. * p ≤ 0.05 relative to A vehicle-treated Pt cells and C Parental NSC (cisplatin-treated) D vehicle-treated Parental or Pt cells as control. Representative blot is shown.