Fig. 5

HPV regulation of CHK1 and Rad51 expression. A HNC cell lines were lysed and analysed by immunoblotting with the indicated antibodies. B Box plot showing means ± SD and individual values of Optical density analysis of the expression of CHK1 and Rad51 normalised to loading control (vinculin) from at least three independent western blot experiments in HNC cell lines. *, P < 0.05; **, P < 0.01 (unpaired t test). C Results from Optical density analysis of SMAD4 in HNC cell lines were correlated to CHK1 and Rad51 and Pearson correlation coefficient (r) was calculated. D, E HPV-positive HNC cell lines were transfected for 72 h with specific siRNA against HPV16 E6/E7 or Luciferase as control. After transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (D) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes of at least three independent experiments (E). F, G HKs were transduced with empty or HPV16 E6/E7 recombinant retroviral vectors. After G418 selection, cells were harvested. F Lysates were collected and analysed by immunoblotting with the indicated antibodies. G Total RNAs were isolated for RT-qPCR. SMAD4 expression was normalized to RpP0. Results from at least three independent experiments are expressed as means ±SD of fold changes. **, P < 0.01; ***, P < 0.001 (unpaired t test). H UMSCC104 cells were transduced with shp53 or control. After antibiotic selection, cells were transfected for 72 h with specific siRNA against HPV16 E6/E7 or Luciferase as control. After transfection cells were lysed and analysed by immunoblotting with the indicated antibodies. I HKs were transduced with empty or HPV16 E6/E7 recombinant retroviral vectors. After G418 selection, cells were seeded on coverslips and after 24 h fixed and stained with the indicated antibodies for Immunofluorescence. Coverslips were mounted and visualized using a Leica SP8 AOBS confocal microscopy