Fig. 4.

The assembly of FANCD2 foci is compromised in FA-J cells. a Levels of endogenous FANCJ were detected with the mouse monoclonal 2G7 antibody in tert-immortalized fibroblasts from a normal control individual (PD846), a FA-A patient (PD845), and two different FA-J patients (EUFA30 and IFAR943/1). The FA-J cells contained vector alone or were corrected by the expression of FANCJ. Expression of FANCJ-Flag-FANCJ in transduced FA-J cell lines was detected with HA.11 mouse anti-HA antibody. Both monoubiquitinated (-L) and unubiquitinated FANCD2 (-S) were detected with E35 antibody. An equivalent amount of protein was loaded from each sample and actin is shown as a loading control. b Examples of FANCD2 foci detected by immunofluorescence microscopy in EUFA30 (FA-J) cells expressing wild-type FANCJ, or containing only the empty pOZ-C vector, or in PD845 (FA-A) cells. An enlargement of the FANCD2-positive cell from the EUFA30+Vector example (cell at upper left) is in the inset for better visualization. Cells were treated with 0.5 μM MMC for 20 h and nuclei were detected by counterstaining with DAPI. c Quantification of the percentage of EUFA30 or IFAR943/1 FA-J cells with FANCD2 foci, either with or without genetic correction. Foci were also quantified in untreated PD845 and PD846 cells, or those exposed to 0.5 μM MMC for 20 h. d Quantification of the percentage of EUFA-30 cells containing empty vector alone, wild-type FANCJ, FANCJ-S990A, or FANCJ-K52R, which displayed FANCD2 nuclear foci, both in untreated populations or following treatment with 0.5 μM MMC for 20 h. Values represent the average of three blind counts of 150 or more cells each±standard deviation from unidentified slides (c–d). For each particular FAJ cell line levels of FANCD2 foci were statistically different (p<0.04) in cells corrected with wild-type FANCJ, as compared with the absence of FANCJ or expression of other forms of the protein (c–d)