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First published online 15 January 2003
doi: 10.1242/jcs.00294

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Nonerythroid II spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links

Department of Pathology and Laboratory Medicine, UMDNJ — New Jersey Medical School and the Graduate School of Biomedical Sciences, Newark, NJ 07103, USA

View larger version (57K): [in a new window]   Fig. 1. Relocalization of SpII* to nuclear foci following treatment of normal cells with 8-MOP plus UVA light. The localization of SpII* in the nucleus of undamaged normal human lymphoblastoid cells or lymphoblastoid cells treated with 8-MOP plus UVA light (6 kJ m-2) was examined by immunofluorescence with purified monoclonal anti--spectrin antibody. Counterstaining for the DNA-specific dye DAPI is shown in the middle panels. In the bottom panels, the fluorescent signal for DAPI was overlaid on that for anti--spectrin. Cells were fixed for staining 15 hours after treatment with 8-MOP plus UVA.

View larger version (55K): [in a new window]   Fig. 2. SpII*, FANCA and XPF co-localize to discrete nuclear foci after treatment of normal cells with 8-MOP plus UVA light. Normal human lymphoblastoid cells were either undamaged or treated with 8-MOP plus UVA light (6 kJ m-2) and the localization of SpII*, FANCA and XPF in the nucleus examined 15 hours after treatment with 8-MOP plus UVA. (A) Dual staining was carried out using affinity-purified monoclonal anti--spectrin antibody and affinity-purified polyclonal anti-FANCA antibody, and stained cells were analyzed by immunofluorescence. When the fluorescent signals for SpII* (green) and FANCA (red) are merged, the overlapping foci are yellow, indicating co-localization of these two proteins. (B) An analysis similar to that in (A) was carried out using anti--spectrin antibody (green) and affinity-purified polyclonal anti-XPF antibody (red). Fluorescent signals for both proteins were merged and overlapping foci appear yellow. (C) Dual staining was also carried out using anti-FANCA (green) and anti-XPF (red) antibodies. The fluorescent signals were merged and overlapping foci are yellow. In all of the above experiments, cells were also stained with the appropriate preimmune sera.

View larger version (20K): [in a new window]   Fig. 3. The number of SpII* nuclear foci increases with increasing dosage of 8-MOP plus UVA light. Formation of SpII* foci in the nuclei of normal human lymphoblastoid cells was examined 15 hours after exposure to varying dosages of 8-MOP plus UVA light. The dosage of 8-MOP plus UVA light was increased by increasing the levels of UVA light at a constant concentration of 8-MOP. Cells stained singly with anti--spectrin were examined. Foci were counted in 100 nuclei at each UVA dose. Nuclei were categorized as having 0-20 foci, 21-40 foci, 41-60 foci, 61-80 foci or 81-100 foci. The y axis indicates the percentage of nuclei in each category. Vertical lines represent s.e.m.

View larger version (28K): [in a new window]   Fig. 4. Formation of SpII*, FANCA and XPF foci in the nucleus depended upon the time after treatment of the cells with 8-MOP plus UVA light. Normal human lymphoblastoid cells were treated with 8-MOP plus UVA light (6 kJ m-2), fixed at the indicated times post-treatment and stained independently using either anti--spectrin, anti-FANCA or anti-XPF antibodies. (A) The percentage of nuclei showing multiple SpII*, FANCA and XPF foci was counted over a period of 0-24 hours after treatment. Nuclei in 100 cells were counted at each time point. (B) The average number of damage-induced SpII*, FANCA and XPF foci per nucleus was counted at time points from 0-24 hours after treatment. In each case, 100 nuclei were counted. Vertical lines indicate ± s.e.m.

View larger version (39K): [in a new window]   Fig. 5. Failure of localization of SpII*, FANCA and XPF to nuclear foci in FA-A cells after treatment with 8-MOP plus UVA light. FA-A (HSC 72) lymphoblastoid cells were either undamaged or treated with 8-MOP plus UVA light (6 kJ m-2) and localization of SpII*, FANCA and XPF in the nuclei examined 15 hours after treatment using immunofluorescence. Cells were in addition stained with the appropriate preimmune serum. (A) Dual staining was carried out using anti--spectrin (green) and anti-FANCA (red) antibodies. Fluorescent signals were not observed for FANCA. Only low levels of SpII* were observed in undamaged nuclei and a small number of SpII* foci were observed in the damaged nuclei. Merged signals show only staining for SpII*. (B) Similar analysis as in (A) was carried out using anti--spectrin (green) and anti-XPF (red) antibodies. Fluorescent signals for both proteins were merged. The yellow dots in the merged images indicate co-localization of both proteins. (C) Dual staining using anti-FANCA (green) and anti-XPF (red) was carried out. Only a few damage-induced foci were observed for XPF in the treated cells and none for FANCA. Merged signals show only staining for XPF.

View larger version (46K): [in a new window]   Fig. 6. In corrected FA-A cells, SpII*, FANCA and XPF co-localize to discrete nuclear foci after DNA cross-link damage. FA-A (HSC 72) cells expressing the FANCA cDNA were either undamaged or treated with 8-MOP plus UVA (6 kJ m-2) and examined for the presence of SpII*, FANCA or XPF in the nuclei. Cells were fixed 15 hours after treatment. (A) Immunofluorescence with anti--spectrin (green) and anti-FANCA (red) antibodies was examined. When the green and red signals were merged, the overlapping foci appeared yellow, indicating co-localization of SpII* and FANCA. (B) An analysis similar to that in (A) was carried out using anti--spectrin (green) and anti-XPF (red) antibodies. The fluorescent signals were merged and analyzed as above. (C) Dual staining with anti-FANCA (green) and anti-XPF (red) antibodies was also examined. Again, the fluorescent signals were merged and overlapping foci appear yellow.

View larger version (31K): [in a new window]   Fig. 7. Binding of SpII*, FANCA and XPF to each other in the nucleus. Chromatin-associated proteins extracts from normal human lymphoblastoid cells were immunoprecipitated with anti--spectrin, anti-FANCA and anti-XPF antibodies. (A) Immunoprecipitation was carried out using anti--spectrin antibody or mouse IgG1 (as a control). (B) Proteins were immunoprecipitated using anti-FANCA antibody or rabbit serum. (C) Immunoprecipitation was done using anti-XPF antibody and mouse serum. In all of the above immunoprecipitations (A-C), immunoblot analysis was carried out using anti-SpII* (top panel), anti-FANCA (second panel) or anti-XPF (third panel).