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. Author manuscript; available in PMC: 2023 Jun 15.
Published in final edited form as: Mol Cell. 2023 Jan 5;83(1):26–42.e13. doi: 10.1016/j.molcel.2022.12.004

Figure 3. Endogenous CDT1 can inhibit DNA synthesis and is counteracted by geminin.

Figure 3

(A and B) RT-QIBC aligned to Sentry(PCNAfoci) in MCF10A cells treated with 2 μM MLN-4924 for 3.5 h during live-cell imaging. Representative of 2 independent experiments. (A) EdU. Error bars are mean ±2 × SEM for each time point (n ≥ 8 cells for all time points, n ≥ 613 cells per condition).

(B) Geminin immunofluorescence 1–3 h after S entry. Box is IQR and median, and whiskers are 1.5 × IQR. G1 cells are DMSO-treated G1 cells (basal geminin), and dashed line is median of G1 cells. n ≥ 37 cells per condition.

(C and D) RT-QIBC in mitogen-released MCF10A cells. MLN-4924 added 4 h before fixation. Cells 2-3 h after APC/CCdh1 inactivation with cyclin E/A-CDK activity ≥0.8.

(C) Dashed lines are negative thresholds, and shaded curves are median in bins of PCNA levels (n ≥ 1,369 cells pooled from 10 replicate wells).

(D) Proportion of PCNA(+) cells that are EdU(−) in each of 10 replicate wells. Error bars are mean ±2 × SEM. Two-sample t test p values: siCtrl DMSO vs. siCtrl MLN-4924 (7.8 × 10−6), siCtrl DMSO vs. siGeminin DMSO (0.38), siCtrl MLN-4924 vs. siGeminin MLN-4924 (2.3 × 10−5).

(E) DNA synthesis is inhibited in the presence of CDT1 after origin firing.

(F-H) RT-QIBC in mitogen-released MCF10A cells 2-3 h after S entry. Representative of 3 independent experiments.

(F) Top:dose-response of EdU to ND-CDT1 expression.Error bars are mean ±2 × SEM for bins of ND-CDT1 expression (bins ≥ 75 cells, 12,039 cells total siCtrl, 4,573 cells total siGeminin). Curves are fit Hill equations. Fit parameters in STAR Methods. Bottom: corresponding cell counts for bins. Dashed lines are endogenous CDT1 levels (CDT1Endogenous) and degraded CDT1 (CDT1 low), calculated from Figure S3H, and fit IC50.

(G) Left: fit ND-CDT1 dose-response curves at 12 levels of geminin expression. Right: color code for geminin expression. GemininLow is negative threshold. n ≥ 660 cells per fit.

(H) Heatmap of median EdU (color) at given geminin and ND-CDT1 levels. Points represent IC50 at each geminin level. (n = 29,350 cells.)

See also Figure S4.