Volume 53, Issue 5, Pages 843-853 (March 2014) Assessing Kinetics from Fixed Cells Reveals Activation of the Mitotic Entry Network at the S/G2 Transition  Karen Akopyan, Helena Silva Cascales, Elvira Hukasova, Adrian T. Saurin, Erik Müllers, Himjyot Jaiswal, Danielle A.A. Hollman, Geert J.P.L. Kops, René H. Medema, Arne Lindqvist  Molecular Cell  Volume 53, Issue 5, Pages 843-853 (March 2014) DOI: 10.1016/j.molcel.2014.01.031 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 A Setup for Quantification of Fluorescence from Single Cells (A) Differential interference contrast (DIC) picture of U2OS cells growing on rectangular micropatterns. (B) Cumulative mitotic entry of U2OS and RPE cells growing either on micropatterns or in a parallel well in the absence of micropatterns. (C) Schematic showing the experimental setup. The coverslip is first scanned using a 10× objective. The positions of single cells on micropatterns are assessed by segmenting images of a DNA-staining and Cy5-labeled fibronectin. Using a 40× objective, the microscope returns to the selected positions and acquires pictures centered on each single cell. Since cells have no neighbors, image segmentation is straightforward. MATLAB scripts for generation of point lists and image segmentation are available in the Supplemental Information. (D) Estimation of a background function. In quantifications of immunofluorescence, additional wells in which primary antibodies are excluded one by one are processed in parallel. The resulting quantification allows estimation of the background as a function of the remaining antibody (red lines). Every dot on the plot represents a single-cell measurement. In addition, for APC/C targets, the second lowest percentile single-cell measurement was subtracted to account for background due to the primary antibody (dotted blue lines, plot on the right). (E) Example of a quantification result. Nuclear cyclin A2 levels versus nuclear DNA. Cytoplasmic cyclin B1 levels versus nuclear DNA. See also Figures S1 and S2. Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Error Estimations on Assessing Cell-Cycle Kinetics from Fixed Cells MATLAB scripts of simulations are available in the Supplemental Information. (A) Schematic of the setup. Cells are ordered after quantification of a protein that increases through the cell cycle. Time is assigned to the first and the last cell and then evenly distributed to all cells in between. (B) Simulation of average error per cell and SD of time estimation for single cells when increasing cell numbers. Cells are ordered based on a hypothetical protein that increases linearly through the cell cycle. Inset: simulation of average error per cell and SD of time estimation for single cells after introducing variation. The impact on assigned time depends on whether the error source is static or related to the levels of the protein being measured. The cell number is fixed to 1,200, and variation is introduced by randomly changing the protein value according to a Gaussian distribution. The SD of the Gaussian distribution is either fixed to the maximal protein value (blue line) or dependent on the protein value (red line). (C) Simulation of time resolution and SD when increasing variation. Time resolution is defined as when the error of the mean of a population equals the time span of that population. Variation is introduced as in (B), where the SD of the Gaussian distribution is fixed to the maximal protein value, corresponding to the blue line in (B). Insets: measured experimental variation from cells growing on micropatterns. Red line indicates a Gaussian distribution with the corresponding SD. (1) 4N peak of DNA staining. (2) Distribution of cyclin A2 signal intensities for a certain range of cyclin B1 intensities measured by immunofluorescence. (3) Distribution of cyclin B1-YFP intensities of live cells measured at the last picture before mitosis (30 min between images). (4) Variation of time from the first to the second mitotic division for U2OS cells. Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Assessing Kinetics from Fixed Cells (A) Quantification of DNA content (DAPI) versus estimated time. Cells were sorted for DAPI, cyclin A2, and cyclin B1 levels. The green and red dotted lines indicate the approximate borders of G1, S, and G2 phases. The cell-cycle phases are based on the DAPI staining in each experiment and are estimated as shown in Figure S4A. (B) Cell-cycle profile of cyclin E (top; cells sorted for DAPI and cyclin B1), cyclin A (middle; cells sorted for DAPI and cyclin B1), and cyclin B (bottom; cells sorted for DAPI and cyclin A2). Mitotic cells (red) are manually added for comparison. (C) Example of cyclin B1-YFP- and cyclin A2-YFP-expressing U2OS cells growing on a micropattern. Top: DIC. Middle and bottom: YFP fluorescence. (D) Quantification of cyclin A2-YFP and cyclin B1-YFP fluorescence of live cells growing on micropatterns. Graphs show average and SD of 13 cyclin A2-YFP-expressing U2OS cells and 17 cyclin B1-YFP-expressing U2OS cells that are synchronized in mitosis (t = 0) in silico. (E) Comparison of quantification of cyclin A2 and cyclin B1 levels using fixed U2OS cells (blue, Figure 3B) and cells expressing cyclin A2-YFP or cyclin B1-YFP (red, Figure 3D). See also Figure S3 and Table S2. Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Cdk1 Target Phosphorylation Appears at the S/G2 Transition All quantifications in this figure are nuclear measurements. (A) Quantification of Aurora A levels versus estimated time. Cells sorted for DAPI and cyclin B1. (B) Quantification of Plk1 levels versus estimated time. Cells sorted for DAPI and cyclin A2. (C) Background subtraction for Cdk1 phosphotargets. Cells were treated with RO-3306 for 2 hr before fixation. After correction for the cell with the second lowest percentile (Figure 1D) and sorting for DAPI and cyclin B1, the resulting staining was used to derive a background function. (D) Quantification of lamin A/C pS22 versus estimated time. Cells sorted for DAPI and cyclin B1. (E) Quantification of cyclin B1 pS126 versus estimated time. Cells sorted for DAPI and cyclin B1. (F) Quantification of lamin A/C pS22 nuclear fluorescence intensity of interphase 4N DNA content cells in the absence of micropatterns. The cytoplasmic staining is used as a background for each cell. Cells were transfected with siRNA for cyclin A2, cyclin B1, or both cyclin A2 and cyclin B1. The box plots represent the 90th, 75th, 50th, 25th, and 10th percentiles of more than 1,000 cells per condition. Black circles represent average intensities. ∗∗∗p < 0.01, Student’s t test. (G) Cells were seeded on micropatterns in the presence of thymidine and fixed after 9 hr. Graph shows quantification of lamin A/C pS22 after sorting for DAPI and Plk1. Note that due to the thymidine treatment, time cannot be estimated. See also Figure S4 and Table S2. Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Plk1 Activity Is First Detected at the S/G2 Transition (A) Quantification of inverted CFP-YFP FRET ratio and area of Tag-RFP foci of U2OS cells coexpressing a probe to monitor the phosphorylation of a Plk1 substrate and a Tag-RFP-labeled chromobody that can bind to PCNA. Nine cells are normalized for maximal area of Tag-RFP foci and synchronized in silico at the time when PCNA foci area starts declining. (B) Example of a time-lapse sequence of a cell quantified in (A) showing a false-colored FRET ratio (top) and Tag-RFP chromobody localization (bottom). The time relative to mitosis is indicated above the images (hr). (C) Quantification of the dynamics of PCNA foci and Plk1 activity in the presence or absence of thymidine. Cells containing PCNA foci at t = 0 were followed as in (B); thymidine was added 1 hr before t = 0. Middle panels: black and red bars show the presence of multiple strong PCNA foci. Gray bars show the presence of few weak PCNA foci that remain after a major decrease in amount and level of PCNA foci occurred. Left (a–d; control) and right (a–h; thymidine) panels show quantifications of inverted FRET ratio and area of Tag-RFP foci for individual cells. Letters indicate the corresponding cell (red bar) in the middle panels. The area of PCNA foci was measured by thresholding for the intensity of the late Tag-RFP foci. (D) Plk1 localization is a marker for Plk1 activity. Images show deconvolved maximum intensity projections of Plk1 and CREST stainings 2 hr after addition of DMSO or the Plk1 inhibitor BI2536. (E) Quantification of relative accumulation of Plk1 to kinetochores and centrosomes. Left: mask created for quantification of Plk1 signal on kinetochores or centrosomes, centered on maximum intensity of the signal (red, 3 × 3 pixels). The background, including signal from soluble Plk1, is estimated in a region surrounding the mask (green, 1 pixel wide). Top right: quantification of Plk1 accumulation on kinetochores versus estimated time. Bottom right: quantification of Plk1 accumulation on centrosomes versus estimated time. See also Figure S5 and Table S2. Molecular Cell 2014 53, 843-853DOI: (10.1016/j.molcel.2014.01.031) Copyright © 2014 Elsevier Inc. Terms and Conditions