Extended Data Fig. 11. Analyses of nuclear CDK1 during cell cycle progression.

a, Immunostaining of human mammary epithelial MCF10A cells treated with control siRNA (siCtrl) or anti-CDK1 siRNA (siCDK1) for 26 h, stained with anti-CDK1 antibody #1 (Abcam). b-d, MCF10A cells expressing Gem^(1–110)-mVenus and Cdt1^{(1–100)Cy(−)}-mCherry FUCCI(CA) reporters were live-imaged and then fixed. Cells were stained with three different anti-CDK1 antibodies: (b) #1 from Abcam, (c) #2 from Atlas Antibodies, (d) #3 from Cell Signaling Technology (CST), and nuclear CDK1 staining was assessed. Upper rows, log₂ intensity of nuclear CDK1 staining at the indicated times after Cdt1^{(1–100)Cy(−)}_drop (S phase start); lower panels, CDK1 nuclear intensity after the end of last mitosis. For control of antibody specificity, cells were transfected with anti-CDK1 siRNA (siCDK1), or control siRNA (siCtrl) for 6 h and then immediately imaged for 16 h. Cells depleted of CDK1 for this time retained sufficient amounts of CDK1 to progress through mitosis. Red lines represent median values within bins every 3 time-points (36 min, 12 min interval). Third panels from left show median log₂ intensity of nuclear CDK1 staining in control cells (blue) and cells treated with anti-CDK1 siRNA (red). Note decreased nuclear CDK1 staining (using all three antibodies) in cells transfected with anti-CDK1 siRNA, which confirms the specificity of the antibodies. Right panels (images of cells) depict examples of nuclear CDK1 staining used for quantification in the previous panels. Upper panels (set of 4 pictures labelled ‘early S phase’), Hoechst (DNA) and CDK1 staining for cells fixed 48 min (4 time-points) after Cdt1^{(1–100)Cy(−)}_drop (S phase start); lower panels labelled ‘G1phase’, cells fixed 3 h after the end of last mitosis in G1. Cells were transfected with control siRNA or anti-CDK1 siRNA. CDK1 depletion (siCDK1) decreased nuclear CDK1 staining in G1 and early S phase cells. e-g, Another representation of values from b-d, for cells in G1 (3–4 h after mitosis) and early S phase (0–1 h after Cdt1^{(1–100)Cy(−)}_drop), transfected with control siRNA (siCDK1 −) or with anti-CDK1 siRNA (siCDK1 +) and stained with anti-CDK1 antibody #1 (e), #2 (f) or #3 (g). Dots show values for individual cells, horizonal lines median values, dotted lines above and below the median represent inter-quartile range. h-i, Live and fixed-cell analysis of primary human dermal fibroblasts, similar to b-d. h, Since cells did not express FUCCI reporters, we analyzed nuclear CDK1 staining relative to the end of last mitosis (left panel). Right panels (cell images) show nuclear CDK1 staining in cells stained 3 h after the end of last mitosis (labelled G1 phase). Nuclei were stained with Hoechst. i, log₂ of EdU incorporation in cells from the left panel (to illustrate their cell cycle progression), pulsed with EdU for 8 min prior to fixation; EdU-high cells correspond to S phase cells, EdU-low to G1 or G2 cells. h-i, Red lines represent median values within bins every 6 time-points (60 min, 10 min interval). Scale bars, 100 μm (a); 10 μm (b-d, h).