Figure 3

The improved-AID system ( i -AID system) promotes efficient Mcm4-aid degradation resulting in acute defect in DNA replication and cell cycle arrest. (A) mcm4-aid cells expressing AtTIR1 under control of the P _nmt41 promoter and Skp1-AtTIR1-NLS under control of the P _nmt41 , P _adh15 or P _adh81 promoter located on the chromosome were spotted onto EMM plates containing auxin (0.5 mM) and incubated at 25°C. (B) mcm4-aid cells harboring the pREP41D-Skp1-AtTIR1 plasmid (MKF60) and those carrying P _nmt41 -skp1-AtTIR1-NLS (HM2491), P _adh15 -skp1-AtTIR1-NLS (HM2473) and P _adh81 -skp1-AtTIR1-NLS (HM2475) on the chromosome were grown in EMM, and the amount of TIR1 in the extracts was analyzed by 7.5% polyacrylamide gel electrophoresis followed by immunoblotting with anti-myc (TIR1) and anti-TAT1 (tubulin) antibodies. (C) Whole-cell extracts of mcm4-aid P _adh15 -skp1-AtTIR1-NLS cells (HM2473) were prepared every 1 h after addition of auxin (0.5 mM) at 25°C. Proteins in whole-cell extracts were separated in 7.5% polyacrylamide gel and analyzed by immunoblotting with anti-Mcm4, anti-myc (TIR1), and anti-TAT1 (tubulin) antibodies. (D) The amount of Mcm4-aid protein after depletion was compared with that in the 0 h sample diluted to 50%, 25%, 12.5% and 6.25%. (E) Whole-cell extracts of mcm4-aid P _adh15 -skp1-AtTIR1-NLS cells (HM2473) were prepared at indicated time point after addition of auxin at 25°C and proteins were analyzed as in (C). (F) DNA contents of HM2473 mcm4-aid P _adh15 -skp1-AtTIR1-NLS cells in the absence (left) or presence (right) of auxin were analyzed by flow cytometry. (G) Relative numbers of HM2473 mcm4-aid P _adh15 -skp1-AtTIR1-NLS cells with or without auxin were measured at the indicated time points by Sysmex (Sysmex Co., Kobe, Japan) [46].