High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
Fig 4
HPV oncogenes increase FancD2 foci formation but impair colocalization of FancD2 with DSBs, and mislocalized FancD2 in E6 cells causes reduction in Rad51 recruitment to DSBs.
(A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.