Figure 1.
Generation of an AID cellular system to assay for mitotic functions of Cdt1 (AID-Cdt1). (A) A schematic of the repair template generated to endogenously tag the Cdt1 genomic locus with YFP and AID at the C-terminal using CRISPR/CAS9-based genome editing. (B) Western-blot analysis using an anti-Cdt1 antibody showing the levels of the endogenous and tagged Cdt1 in parental and AID-Cdt1 clones, respectively, following auxin-mediated degradation. (C) AID-Cdt1 DLD1 cells growing at 37°C were treated with either control DMSO (top panel, [−] IAA) or the Auxin, IAA, for 2 h. (middle and bottom panels, [+] IAA) followed by immunostaining using an antibody against tubulin and the chromosomes counterstained with DAPI. Scale bar, 5 µm. (D) AID-Cdt1 DLD1 cells growing at 37°C were treated with the DNA stain, Hoechst, along with either control DMSO (top panels) or IAA, for 2 h. (middle and bottom panels) followed by live-cell imaging for 2–6 h as required. Selected frames from the time series are shown for each experimental condition as indicated. Scale bar, 5 µm. (E) Quantification of live imaging data from D. n = 30 for control cells not treated with IAA and 55 for cells treated with IAA. Data presented as mean ± SD, two-tailed t test *** = P < 0.0001. (F) AID-Cdt1 DLD1 cells growing at 37°C were treated with either control DMSO (top panel) or IAA, for 2 h (bottom panel) followed by treatment with ice-cold buffer PBS buffer for 10 min. The cells were then immunostained for antibodies against tubulin, Zwint1 (for kinetochores) with the chromosomes counterstained using DAPI. Scale bar, 5 µm. (G) Quantification of total microtubule intensities from cells in F (n = 10 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (H) Insets from the top and bottom panels of F showing samples of attached and unattached kinetochores, respectively, as indicated. Scale bar, 1 µm. (I) Quantification of the % of attached kinetochores from F under the two experimental conditions assessed (n = 10 cells). Data presented as mean ± SD. Source data are available for this figure: SourceData F1.

Generation of an AID cellular system to assay for mitotic functions of Cdt1 (AID-Cdt1). (A) A schematic of the repair template generated to endogenously tag the Cdt1 genomic locus with YFP and AID at the C-terminal using CRISPR/CAS9-based genome editing. (B) Western-blot analysis using an anti-Cdt1 antibody showing the levels of the endogenous and tagged Cdt1 in parental and AID-Cdt1 clones, respectively, following auxin-mediated degradation. (C) AID-Cdt1 DLD1 cells growing at 37°C were treated with either control DMSO (top panel, [−] IAA) or the Auxin, IAA, for 2 h. (middle and bottom panels, [+] IAA) followed by immunostaining using an antibody against tubulin and the chromosomes counterstained with DAPI. Scale bar, 5 µm. (D) AID-Cdt1 DLD1 cells growing at 37°C were treated with the DNA stain, Hoechst, along with either control DMSO (top panels) or IAA, for 2 h. (middle and bottom panels) followed by live-cell imaging for 2–6 h as required. Selected frames from the time series are shown for each experimental condition as indicated. Scale bar, 5 µm. (E) Quantification of live imaging data from D. n = 30 for control cells not treated with IAA and 55 for cells treated with IAA. Data presented as mean ± SD, two-tailed t test *** = P < 0.0001. (F) AID-Cdt1 DLD1 cells growing at 37°C were treated with either control DMSO (top panel) or IAA, for 2 h (bottom panel) followed by treatment with ice-cold buffer PBS buffer for 10 min. The cells were then immunostained for antibodies against tubulin, Zwint1 (for kinetochores) with the chromosomes counterstained using DAPI. Scale bar, 5 µm. (G) Quantification of total microtubule intensities from cells in F (n = 10 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (H) Insets from the top and bottom panels of F showing samples of attached and unattached kinetochores, respectively, as indicated. Scale bar, 1 µm. (I) Quantification of the % of attached kinetochores from F under the two experimental conditions assessed (n = 10 cells). Data presented as mean ± SD. Source data are available for this figure: SourceData F1.

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