Cells were transfected with non-targeting (NT) or HERC1 siRNA. For U2OS cells, two different HERC1 siRNAs (Q1 and Q4) were used. (A, D) Seventy-two hours later, lysates were analyzed by immunoblotting with antibodies against the indicated proteins. (B, C, E, F) Colony formation assays were performed as indicated in “Materials and Methods”. After 12-15 days, cells were stained with crystal violet (B, E). Crystal violet absorbance at 550 nm was quantified (n = 6 for U2OS cells; n = 3 for HeLa cells) (C, F). CHC: Clathrin heavy chain. Data are expressed as mean ± S.E.M. Statistical analysis was carried out as described under “Materials and Methods”. The differences are shown with respect to NT siRNA. ***, p< 0.001.