Figure 1. Generation of a study system with FA-signaling-defective cells. (A) MDA-MB-231 cells do not have the endogenous expression of FANCI, but have a relatively low level of FANCC expression. We examined the expression of FANCI and FANCC by western blotting in MDA-MB-231, HEK 293T, U2OS, CRL-1197, HTB-4, and U251 cells, among which the level of FANCI protein expression is undetectable in MDA-MB-231cells. However, the level of FANCC expression was low compared to the other cells detected. (B) Ectopic expression of FANCC in MDA-MB-231 cells. Cells were transfected with empty vectors for control or plasmids carrying FANCC cDNA. The level of FANCC was detected accordingly by western blotting in pool-selected two groups of cells. These derivatives of MDA-MB-231 express FANCC at a low or high level (namely FANCC-low or -high cells). (C) Number of colonies derived from FANCC-high cells is reduced compared to FANCC-low cells. Colony formation of FANCC-high or low cells was determined by the soft agar assay. The number of colonies was counted and showed significantly different between two groups (p<0.001). (D) FANCC-high cells have a reduced capacity for migration. Both cell images and numbers of migrated cells showed that FANCC can decrease the capacity of MDA-MB231 cell migration. The number of migrated cells was counted and showed significantly different between two groups (p<0.001).