U2-OS cells were transfected with either Control siRNA, or siRNA against FancI (A,B), or FancJ (C–E) and then split into three sets. One set of cells was used for Western Blot analysis to monitor the siRNA knockdown efficiency. Antibodies used for immunoblotting are indicated on the right (A,C). The second set of cells was fixed in methanol and stained with antibody against γ-Tubulin (B,D). The third set of cells was first treated with 16 mM HU for 68 hours and then fixed in methanol and stained with antibody against γ-Tubulin (B,E). More than 300 cells were counted and the percentage of cells with more than two centrosomes was quantitated (B,D,E). * indicates the band corresponding to FancI. Mock refers to cells without siRNA transfection. All error bars are standard deviation obtained from three different experiments. Standard two-sided t test, *P<0.05, **P<0.01, ***P<0.001.