Co-immunofluorescence microscopic analysis of U-2 OS cells stably expressing HPV-16 E7 for FANCD2 in combination with RPA32, BLM, RAD51, MRE11, BRCA2/FANCD1 or MUS81. MUS81-HA was transiently overexpressed in cells before processing for immunofluorescence staining for HA and FANCD2. Inserts correspond to APBs indicated by arrows. Nuclei stained with DAPI. Scale bar indicates 10 μm.

(A) Co-immunofluorescence microscopic analysis of primary human keratinocytes stably expressing HPV-16 E7 or empty vector control (LXSN) for FANCD2 and TRF2. Arrow indicates the focus shown in the insert. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(B) Quantification of the percentage of primary human keratinocytes stably expressing HPV-16 E7 (closed boxes) or control (LXSN; open circles) that contain FANCD2-positive APBs. Each data point represents mean and standard error of at least triple quantification of a minimum of 500 cells at the passage number indicated.

(A) Co-immunofluorescence microscopic analysis of U-2 OS cells stably expressing HPV-16 E7 for FANCD2 and PML (top panels) or FANCD2 and TRF2 (middle panels). Immunofluorescence analysis for FANCD2 combined with fluorescence in situ hybridization (FISH) using a Cy3-conjugated telomeric probe is shown in the bottom panels. Arrows indicate the foci shown in the inserts. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(B) Co-immunofluorescence microscopic analysis of U-2 OS cells stably expressing empty vector (control) or HPV-16 E7 for BrdU to visualize ssDNA and FANCD2. Arrow indicates the APB shown in the inserts. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(C) Quantification of the proportion of cells with BrdU- and FANCD2-positive APBs in U-2 OS cell populations stably expressing empty vector (control) or HPV-16 E7. Each bar represents mean and standard error of two independent experiments with at least triple quantification of a minimum of 100 cells per experiment.

(A) Immunoblot analysis of U-2 OS cells at 72 h after transient transfection with either small hairpin RNA directed against FANCD2 (shFANCD2) or control (shGFP). Immunoblot for actin is used to demonstrate protein loading.
(B) Immunofluorescence analysis of U-2 OS cells at 72 h after transient transfection with shFANCD2 or control (shGFP) for 53BP1 combined with telomere fluorescence in situ hybridization (tel. FISH) using a Cy3-conjugated telomeric probe. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(C) Quantification of the percentage of U-2 OS cells transiently transfected with shFANCD2 or shGFP that contain more than five telomeric signals that co-localize with 53BP1. Each bar represents mean and standard error of two independent experiments with at least duplicate quantification of a minimum of 50 cells per experiment.

(A) Immunofluorescence microscopic analysis of FANCD2 in U-2 OS cells stably expressing empty vector (control) or the HPV-16 E7 oncoprotein. Note the unusually large and ring-shaped FANCD2 staining (arrow; insert) in comparison to the small punctate staining pattern in the control cell. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(B–D) Quantification of the percentage of cells with ring-shaped FANCD2 foci in U-2 OS cells stably expressing HPV-16 E7 (B), HPV-16 E6 (B), low risk HPV-6 E7 (C) or various mutants of HPV-16 E7 (D). Empty vector transfected cells are shown as control. Expression of HPV proteins was verified in all experiments by immunoblotting or PCR (not shown). Each bar in B represents mean and standard error of at least three independent experiments with at least 100 cells counted per experiment. Each bar in C and D represents an at least triple quantification of a least 100 cells of a representative experiment. In all figures, asterisks are shown to indicate statistically significant differences in comparison to controls (Student’s t test for independent samples).

(A) Quantification of the percentage of U-2 OS cells with FANCD2-positive APBs following treatment with 100 ng/ml MMC or dH₂O (control) for 24 h (left panel) or treatment with 1 mM of hydroxyurea (HU) or dH₂O (control) for the indicated time intervals (right panel). Each bar represents mean and standard error of al least two independent experiments with at least triple quantification of a minimum of 50 cells per experiment.
(C) Immunoblot analysis of U-2 OS cells stably expressing empty vector (control) or HPV-16 E7 after transfection with control siRNA duplexes (ctrl.) or siRNA duplexes targeting ATM or ATR for the indicated time intervals. Immunoblot for actin is shown to demonstrate loading of equal amounts of protein.
(D) Immunofluorescence microscopic analysis of U-2 OS cells stably expressing HPV-16 E7 at 72 h after transfection with control siRNA duplexes or siRNA duplexes targeting ATM or ATR. Note the absence of FANCD2-positive APBs in HPV-16 E7-expressing cells transfected with siRNA duplexes targeting ATR. A DsRED expression plasmid with a mitochondrial target sequence was used as transfection marker. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(E) Quantification of the percentage of HPV-16 E7-expressing U-2 OS cells displaying FANCD2-containing APBs following transfection with the respective siRNA duplexes. Each bar indicates mean and standard error of two independent experiments with at least triple quantification of a minimum of 100 cells per experiment.