Production and characterization of ETAA1 and TOPBP1-deficient cell lines. (A) Schematic of the ETAA1ΔAAD gene and protein. (B) HCT116 and HCT116 ETAA1ΔAAD cells were lysed and immunoblotted with ETAA1 antibodies. ETAA1 electrophoretic mobility is altered following CPT treatment. (C) Schematic of the TOPBP1-AID gene and protein. (D) HCT116 WT and TOPBP1-AID cells were treated with 500 µM IAA for the indicated times. Cells were lysed and immunoblotted with the indicated antibodies. Upper band is full-length TOPBP1-AID, and lower band is TOPBP1. (E–H) HCT116 WT, ETAA1ΔAAD, TOPBP1-AID, and ETAA1ΔAAD/TOPBP1-AID cells were left untreated (E and F) or treated (G and H) with 500 µM IAA. Viable cells numbers were measured by staining with trypan blue. Data points and error bars are mean and standard deviation of three experiments. (I) HCT116 WT, ETAA1ΔAAD, TOPBP1-AID, and ETAA1ΔAAD/TOPBP1-AID cells were left untreated or treated with 500 µM IAA for 2 h before examining cell cycle distribution by flow cytometry. Data points and error bars are the mean and standard deviation of three experiments. n.s., not significant. Molecular weight is given in kilodaltons.