Analyses of the AID-Cdt1 degron system to study mitotic functions of Cdt1. (A) Western blot analysis using an anti-GFP antibody showing the levels of tagged-Cdt1 in parental DLD1 cells and in the YFP-tagged AID-Cdt1 clones following auxin-mediated degradation. (B) Selection of homozygous AID-Cdt1 colonies by PCR. (C) AID-Cdt1 DLD1 cells were treated with the DNA stain, Hoechst, along with either control DMSO (top panel) or IAA (bottom panel) for 2 h and still pictures of the chromosomes and YFP were acquired. (D) AID-Cdt1 DLD1 cells were treated with ice-cold (4°C) buffer for 10 min after adding IAA for the indicated periods of time. The cells were then immunostaining for antibodies against tubulin and the chromosomes counterstained with DAPI. (E) AID-Cdt1 DLD1 cells growing at 37°C were fixed after adding IAA for the indicated periods of time. The cells were then immunostained for antibodies against tubulin and the chromosomes counterstained with DAPI. (F) Parental DLD1 cells treated with DNA damaging agent, Etoposide, for 4 h, control AID-Cdt1 DLD1 cells or those treated with IAA for 2 h were immunostained for antibodies against γ-H2AX and the chromosomes were counterstained with DAPI. (G) Control AID-Cdt1 DLD1 cells or those treated with IAA for 2 h were immunostained for antibodies against microtubules and the chromosomes were counterstained with DAPI. Scale bar, 5 µm. Source data are available for this figure: SourceData FS1.