Fig. 6

Analysis of HPV modulation of DD response upon SMAD4 silencing. A, B HPV-positive (UMSCC104) and HPV-negative (UMSCC18) HNC cell lines were transfected with specific siRNA against SMAD4 or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (A) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes of at least three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (unpaired t test) (B). C, D HKs were transduced with empty or HPV16 E6/E7 recombinant retroviral vectors. After selection with G418 cells were transfected with specific siRNA against SMAD4 or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (C) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (One-way Anova Multicomparison test) (D). E, F UDSCC2 cell line was transfected with specific siRNA against SMAD4 and HPV16 E6/E7 alone or in combination, or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (E) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes over siLUC. *, P < 0.05, ns, not significant (One-way Anova Multicomparison test) (F). G UDSCC2 cell line was transfected with specific siRNA against SMAD4 or Luciferase as control. 48 h after transfection cells were treated for 24 h with 10 μM Cisplatin and subsequently lysed and analysed by immunoblotting with the indicated antibodies. H UDSCC2 cell line was transfected with specific siRNA against SMAD4 or Luciferase as control. 24 h after transfection cells were seeded into 96well plates (7000 cells/well) and 24 h later were treated with 10 μM Cisplatin for 24 h. Cell viability was then assessed using CellTiter-Glo® Luminescent Cell Viability Assay and expressed as viability relative to untreated cells (means ±SD of fold changes) of two independent experiments. *, P < 0.05; (One-way Anova Multicomparison test)