Table 1.
Candidate clones obtained from Yeast-2-hybrid screens.
Table 2.
Yeast-2-hybrid assays between FANCC1-306 and the identified clones.
Figure 1.
FANCC directly interacts with UNC5A in yeasts.
Yeast two-hybrid assays were performed with various UNC5A constructs that included the Y2H clone coding for the C-terminal death domain (DEATH) and a part of intron 14 (shown in blue), UNC5A intracellular domain (UNC5AICD), the UNC5A C-terminus (UNC5ADD) and the death-domain deletion mutant (UNC5AΔDD). The yeast strain AH109 was co-transformed with UNC5A constructs along with FANCC constructs as indicated. The plus sign (+) indicate a positive interaction. Negative controls were performed using empty vectors (Empty), and positive controls were performed using pGBKT7/p53 and pGADT7/SV40 T-antigen from Clontech (data not shown). Yeast two-hybrid assays were performed at least three times (independent transformation) with 3 to 4 clones each.
Figure 2.
Immunoprecipitations (IPs) were performed in HEK293T cells transiently transfected with Myc-tagged rUNC5A (A), HA-tagged UNC5AICD (B–C), with (A–B) or without (C) full-length FANCC. In D and E, HEK293T cells were cotransfected with HA-tagged UNC5AICD and EGFP-tagged FANCC1-306 (D) or EGFP-tagged FANCC307-558) (E). IPs were performed using anti-FANCC, anti-Myc, anti-HA, anti-GFP or control mouse serum (IgG). Western immunoblotting (IB) was performed with the indicated antibodies. WCE: whole cell extract from transfected cells, C: control untransfected cells. * indicates non-specific or IgG bands. Numbers indicate molecular weight.
Figure 3.
FANCC interacts with UNC5AΔDD.
Immunoprecipitations (IPs) were performed in HEK293T cells transiently transfected with Xpress-tagged death-domain deletion mutant UNC5A (UNC5AΔDD) and HA-tagged FANCC (A) or in B HA-tagged FANCC harboring the L554P mutation (HA-FANCCL554P). IPs were performed using anti-Xpress, anti-HA or control mouse serum (IgG). Western immunoblotting (IB) was performed with the indicated antibodies. WCE: whole cell extract from transfected cells. * indicates non-specific or IgG bands. Numbers indicate molecular weight
Figure 4.
FANCC co-localizes with UNC5A in the cytoplasm.
Representative immunofluorescence experiment performed in HEK293T cells double-stained with anti-UNC5A (red) and anti-FANCC (green) antibodies. Cells were visualized via confocal fluorescence microscopy using a Nikon E800 microscope equipped with a C1 confocal system at 100X magnification.
Figure 5.
FANCC interferes with UNC5A-mediated apoptosis.
HeLa cells were transiently transfected with EGFP-UNC5AICD with or without full-length FANCC or empty vectors. At 48 hours after transfection, cells were fixed and labeled with anti-cleaved-caspase-3 antibodies, and visualized by fluorescence microscopy using a Nikon E800 microscope equipped with a C1 confocal system at 60X magnification. (A) Representative immunofluorescence experiment showing EGFP-UNC5AICD transfected cells (green) and cleaved-caspase-3 positive cells (red). (B) Data are expressed as the mean percent ± standard error of the mean (SEM) of cleaved-caspase-3-positive cells out of EGFP-positive cells from 3 separate experiments. (C) Data are expressed as ratio of caspase-3-positive cells obtained from EGFP-positive UNC5AICD cells compared to cells transfected with empty vectors.
Figure 6.
FANCC-depleted cells are sensitive to UNC5A-mediated apoptosis.
SH-SY5Y cells were stably transduced with shRNA against FANCC (sh-C1 and sh-C2) or scrambled shRNA vectors (C). FANCCi and cells transduced with scrambled shRNA (control) were transfected with UNC5AICD or an empty vector. At 48 hours after transfection, cells were fixed and labeled with anti-cleaved-caspase-3 antibodies, and analyzed by fluorescence microscopy. Data are expressed as the mean cleaved-caspase-3 positive cells ± SEM from three separate experiments performed in duplicate. P values are indicated in the graph.
Figure 7.
Schematic representation of FANCC interaction with UNC5A and possible role in apoptosis.
Based on interactions studies, full length FANCC interacts with UNC5A via different parts of the intracellular domain. Upon apoptosis, FANCC and UNC5A are cleaved by a caspase. Cleaved FANCC fragments interact with UNC5A and interfere with the apoptosis signal (survival). In absence of FANCC or mutations in FANCC, UNC5A triggers apoptosis.