Figure 3.
De novo CENP-A deposition during quiescence exit occurs following the first mitosis. (A) Diagram showing experimental conditions for GFP-CENP-A quiescence exit live imaging experiments from Fig. 3, D–F. (B) Representative images of live GFP-CENP-A–expressing cells imaged for GFP from three different conditions: asynchronous cycling, 7 days of quiescence, or 12 h after release from quiescence. Scale bar = 10 µm (C) Graph quantifying cells from experiment in Fig. 3 B. GFP centromere intensity was measured in GFP-CENP-A–expressing cells from different conditions: asynchronous cycling, 7 days of quiescence, or 12 h after release from quiescence. Cells were imaged live for GFP. Each point indicates the average centromere intensity level for 10 centromeres in a single cell, adjusted for background. Red line represents the median, and blue points represent averages for each replicate. Three replicates were conducted. Intensity values are normalized to cycling. P = 0.0054 between cycling and quiescent, P = 0.0026 between cycling and 12 h release, and P = 0.5430 between quiescent and 12 h release. ** represents P < 0.01; ns is not significant. n = 232, 256, 237 cells for cycling, quiescent, and 12 h release, respectively. P values calculated using unpaired t test with Welch’s correction. (D) Graph showing the normalized level of GFP-CENP-A intensity at the centromeres as cells exit quiescence. Red arrow indicates time of mitosis. X-axis spans from 12 h before and 12 h after mitosis. Black line shows the means, and green shows mean ± SD. Data are aggregated from 33 cells quantified over 15-min or 20-min timeframes from 3 separate biological replicates. (E) Graph showing GFP-CENP-A intensity at the centromeres for cells released from quiescence before and after their first mitosis and for cycling cells (from Fig. 3 D, three replicates). Pre-mitosis values are the average of all measurements from beginning of the live imaging or time of cell entry into imaging area to time of mitosis. Post-mitosis values are the average of all measurements from 1 h after the end of mitosis to the end of the time course or exit of the cell from the imaging area. Cycling values are from asynchronous control cells from the last imaging time frame. Each point indicates the average centromere intensity level for 10 centromeres or max number visible in a single cell, adjusted for background. Red line represents the median, and blue points are the average of each replicate. Intensity values are normalized to cycling. * represents P < 0.05, ** represents P < 0.01, and ns = not significant. P = 0.0427 between before mitosis and after mitosis, P = 0.0068 between before mitosis and cycling, and P = 0.6628 between after mitosis and cycling. P values were calculated using unpaired t test with Welch’s correction. (F) Live imaging stills of a representative GFP-CENP-A cell exiting quiescence. Entry into mitosis is indicated. After mitosis, both cells are indicated and shown. Time units are hours:minutes. (G) Western blot of 7-day quiescent cells, cells released for 16 h from quiescence (G1), asynchronous cycling cells, cells arrested in G2 using RO-336 (Vassilev, 2006; Vassilev et al., 2006), and cells arrested in mitosis using S-trityl-L-cysteine (STLC) (Skoufias et al., 2006). Blot was incubated with cyclin B1 antibody, and α-tubulin is used as a loading control. (H) Representative immunofluorescence images of cycling cells in G1 (identified by presence of a midbody), 7-day quiescent cells, and cells released from quiescence for 16 h stained for PLK1, and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (I) Graph representing PLK1 intensity levels at the centromere for experiment from Fig. 3 H. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling G1. Red line represents the median, and blue points represent average of each replicate. **** represents P < 0.0001, using unpaired t test with Welch’s correction. Source data are available for this figure: SourceData F3. Refer to the image caption for details. Panel A: Diagram showing experimental conditions for G F P-C E N P-A quiescence exit live imaging experiments. The timeline shows a 7-day serum starvation, a release, 12 hours leading to the start of imaging, and a 36-hour observation period, mapping the experiment's chronological progression. Panel B: Representative images of live G F P-C E N P-A-expressing cells under three conditions: asynchronous cycling, 7 days of quiescence, or 12 hours after release from quiescence. Scale bar equals 10 micrometers. Panel C: Box plot quantifying G F P centromere intensity in cells from different conditions. The horizontal axis shows conditions (cycling, quiescent, 12-hour release), and the vertical axis shows normalized G F P-C E N P-A intensity. The red line represents the median, and the blue points represent averages for each replicate. Panel D: Line graph showing normalized G F P-C E N P-A intensity at the centromeres as cells exit quiescence. The horizontal axis spans from 12 hours before and 12 hours after mitosis, and the y-axis shows normalized G F P-C E N P-A intensity. The black line shows the mean, and the green line shows the mean plus or minus standard deviation. Panel E: Box plot showing G F P-C E N P-A intensity at the centromeres for cells released from quiescence before and after their first mitosis and for cycling cells. The horizontal axis shows conditions (pre-mitosis, post-mitosis, cycling), and the vertical axis shows normalized G F P-C E N P-A intensity. The red line represents the median, and the blue points represent averages for each replicate. Panel F: Live imaging stills of a representative G F P-C E N P-A cell exiting quiescence, indicating entry into mitosis and post-mitosis cells. Time units are hours: minutes. Panel G: Western blot showing Cyclin B 1 levels in different cell cycle stages, with alpha-tubulin as a loading control. Panel H: Immunofluorescence images of cycling cells in G 1, 7-day quiescent cells, and cells released from quiescence for 16 hours, stained for P L K 1 and anti-centromere (A C A) antibodies. Scale bar equals 5 micrometers. Panel I: Box plot representing P L K 1 intensity levels at the centromere for the experiment in Panel H. The horizontal axis shows conditions (cycling G 1, quiescent, release G 1), and the vertical axis shows normalized P L K 1 intensity. The red line represents the median, and the blue points represent averages for each replicate. All data is approximate.

De novo CENP-A deposition during quiescence exit occurs following the first mitosis. (A) Diagram showing experimental conditions for GFP-CENP-A quiescence exit live imaging experiments from Fig. 3, D–F. (B) Representative images of live GFP-CENP-A–expressing cells imaged for GFP from three different conditions: asynchronous cycling, 7 days of quiescence, or 12 h after release from quiescence. Scale bar = 10 µm (C) Graph quantifying cells from experiment in Fig. 3 B. GFP centromere intensity was measured in GFP-CENP-A–expressing cells from different conditions: asynchronous cycling, 7 days of quiescence, or 12 h after release from quiescence. Cells were imaged live for GFP. Each point indicates the average centromere intensity level for 10 centromeres in a single cell, adjusted for background. Red line represents the median, and blue points represent averages for each replicate. Three replicates were conducted. Intensity values are normalized to cycling. P = 0.0054 between cycling and quiescent, P = 0.0026 between cycling and 12 h release, and P = 0.5430 between quiescent and 12 h release. ** represents P < 0.01; ns is not significant. n = 232, 256, 237 cells for cycling, quiescent, and 12 h release, respectively. P values calculated using unpaired t test with Welch’s correction. (D) Graph showing the normalized level of GFP-CENP-A intensity at the centromeres as cells exit quiescence. Red arrow indicates time of mitosis. X-axis spans from 12 h before and 12 h after mitosis. Black line shows the means, and green shows mean ± SD. Data are aggregated from 33 cells quantified over 15-min or 20-min timeframes from 3 separate biological replicates. (E) Graph showing GFP-CENP-A intensity at the centromeres for cells released from quiescence before and after their first mitosis and for cycling cells (from Fig. 3 D, three replicates). Pre-mitosis values are the average of all measurements from beginning of the live imaging or time of cell entry into imaging area to time of mitosis. Post-mitosis values are the average of all measurements from 1 h after the end of mitosis to the end of the time course or exit of the cell from the imaging area. Cycling values are from asynchronous control cells from the last imaging time frame. Each point indicates the average centromere intensity level for 10 centromeres or max number visible in a single cell, adjusted for background. Red line represents the median, and blue points are the average of each replicate. Intensity values are normalized to cycling. * represents P < 0.05, ** represents P < 0.01, and ns = not significant. P = 0.0427 between before mitosis and after mitosis, P = 0.0068 between before mitosis and cycling, and P = 0.6628 between after mitosis and cycling. P values were calculated using unpaired t test with Welch’s correction. (F) Live imaging stills of a representative GFP-CENP-A cell exiting quiescence. Entry into mitosis is indicated. After mitosis, both cells are indicated and shown. Time units are hours:minutes. (G) Western blot of 7-day quiescent cells, cells released for 16 h from quiescence (G1), asynchronous cycling cells, cells arrested in G2 using RO-336 (Vassilev, 2006; Vassilev et al., 2006), and cells arrested in mitosis using S-trityl-L-cysteine (STLC) (Skoufias et al., 2006). Blot was incubated with cyclin B1 antibody, and α-tubulin is used as a loading control. (H) Representative immunofluorescence images of cycling cells in G1 (identified by presence of a midbody), 7-day quiescent cells, and cells released from quiescence for 16 h stained for PLK1, and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (I) Graph representing PLK1 intensity levels at the centromere for experiment from Fig. 3 H. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling G1. Red line represents the median, and blue points represent average of each replicate. **** represents P < 0.0001, using unpaired t test with Welch’s correction. Source data are available for this figure: SourceData F3.

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