Figure 3.

Overexpression of missense variants in FANCB-null fibroblasts show varying degree of residual function. hemagglutinin (HA)-tagged complementary DNA of FANCB WT or 5 missense variants were overexpressed by lentiviral vector in FANCB-null fibroblasts, followed by functional analysis. (A) Western blot of FANCD2, tubulin, and HA-FANCB. Near-normal FANCD2 ubiquitination was observed upon mitomycin C (MMC) exposure in cells expressing p.W479G and p.L676P, whereas cells expressing p.L43S showed almost no FANCD2 ubiquitination. Expression of p.L329P and p.G750V resulted in low level of FANCD2 ubiquitination. HA-FANCB p.G750V was unstable. Tubulin was used as loading control. Relative ratio of ubiquitinated FANCD2 band to nonubiquitinated FANCD band was measured for each variant. (B) Immunofluorescence of FANCD2 and HA-FANCB. Cells expressing HA-FANCB missense variants displayed smaller and fewer FANCD2 foci than cells expressing WT FANCD2 (original magnification ×630 oil immersion). (C) Quantification of FANCD2 foci–positive cells. One hundred cells were counted in triplicate per each experiment. Three independent experiments were performed. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. (D) Fibroblasts expressing FANCB WT or missense variants were treated with corresponding doses of MMC. Surviving cells were counted, and survival was calculated relative to untreated cells. Three independent experiments were performed. A graph from a representative experiment is shown. *P < .05, ****P < .001. DAPI, 4′,6-diamidino-2-phenylindole; EV, empty vector; ns, not significant.