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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: HPV-mediated regulation of SMAD4 modulates the DNA damage response in head and neck cancer

Fig. 3

HPV16 regulation of SMAD4 degradation. A HPV-negative (UMSCC4, UMSCC6, UMSCC10A and UMSCC18) and HPV-positive (UMSCC47, UMSCC104, UPCISCC154, UDSCC2 and 93VU147T) HNC cell lines were treated with 10 μM Cycloheximide (CHX) for 16 h, lysed and analysed by immunoblotting. Results are represented as Box plot showing percentage of remaining protein ± SD after CHX treatment, calculated from Optical density analysis of the expression of SMAD4 normalized to loading control. *, P < 0.05 (unpaired t test). B UPCISCC154 were transfected with specific siRNA against HPV16 E6/E7 or Luciferase as control for 72 h and treated with two concentrations (3 or 5 μM) of MG132. Cells were then lysed and analysed by immunoblotting with the indicated antibodies. C HPV-positive (UPCISCC154 and 93VU147T) and HPV-negative (UMSCC10A and UMSCC18) HNC cell lines were transfected with specific siRNA against βTRCP1 or Luciferase. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies. D Graph shows the Optical density (O.D.) analysis of the expression of SMAD4 normalised to loading control (vinculin) and expressed as means ±SD of fold changes. *, P < 0.05 (Two-way Anova Multicomparison test) (E) HNC HPV-positive cell lines were transfected with specific siRNA against HPV16 E6/E7 or Luciferase as control. 72 h after transfection total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes. *, P < 0.05 (unpaired t test) (F) UMSCC104 cell line was transfected with specific siRNA against HPV16 E6/E7 or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies. G HKs were transduced with empty or HPV16 E6/E7 recombinant retroviral vectors. After selection with G418 cells total RNAs were isolated for RT-qPCR. βTRCP1 expression was normalized to RpP0. Results from at least three independent experiments are expressed as means ±SD of fold changes. ****, P < 0.0001 (unpaired t test)

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