Figure S1.
Controls for quiescence induction and conservation of quiescent centromere behavior in mouse cells. (A) Representative images of EdU staining of cycling and quiescent cells. Cells were incubated for 48 h in EdU, a nucleotide analog that monitors DNA replication and progression through the cell cycle. Scale bar = 100 µm. (B) Graph showing the percentage of EdU-positive cells for the indicated condition. Cells were incubated for 48 h in EdU. Bars represent mean ± SD of three replicates. Mean of cycling is 95.12; mean of quiescence is 0.2. ** represents P = 0.0015, using unpaired t test with Welch’s correction. (C) Histogram showing the distribution of PI staining for cycling cells (blue) or cells in quiescence for 7 days (green) as measured by flow cytometry. (D) Graph showing CENP-A centromere intensity level over time of quiescence entry. Each point indicates the average centromere intensity level for all centromeres of a single cell, adjusted for background. Intensity values were normalized to day 0. Red line represents the median, and blue points represent average of each replicate. Points were from aggregated from six replicates. n = 440, 661, 768, 726, 686, and 555 cells for 0, 1, 2, 3, 5, and 7-day time points, respectively. * represents P < 0.05; P = 0.0297 between cycling and 7-day quiescent, averages were 1 and 0.8429, respectively. P values were calculated using unpaired t test with Welch’s correction. (E) Graph showing CENP-T centromere intensity levels in cycling and quiescent mouse 3T3 cells. Cells were quiescent for 7 days. Each point indicates the average centromere intensity level for a single cell, adjusted for background. Intensity values were normalized to cycling condition. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 206 and 223 cells for cycling and quiescent, respectively. P = 0.0031, using unpaired t test with Welch’s correction. (F) Representative immunofluorescence images of cycling and 7-day quiescent mouse 3T3 cells. Cells were stained with mouse CENP-T and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (G) Graph showing CENP-C centromere intensity levels in cycling and quiescent mouse 3T3 cells. Cells were quiescent for 7 days. Each point indicates the average centromere intensity level for a single cell, adjusted for background. Intensity values were normalized to cycling condition. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 195 and 254 cells for cycling and quiescent, respectively. P = 0.0342, using unpaired t test with Welch’s correction. (H) Representative immunofluorescence images of cycling and 7-day quiescent mouse 3T3 cells. Cells were stained with mouse CENP-C and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (I) Western blot of cells treated with 50 nM CENPT or control siRNAs. Blot verifies the banding pattern for CENP-T antibody. CENP-T is the upper band. Blot was incubated in CENP-T antibody. (J) Western blot of cells treated with 50 nM CENPC or control siRNAs. Blot verifies the banding pattern for CENP-C antibody. Blot was incubated in CENP-C antibody. (K) Western blot of cells treated with 50 nM CENPH or control siRNAs. Blot verifies the banding pattern for CENP-H antibody. CENP-H is the upper band. Blot was incubated in CENP-H antibody. Source data are available for this figure: SourceData FS1. Refer to the image caption for details. Panel A: Two sets of images show E d U staining of cycling and quiescent cells. The cycling cells exhibit significant E d U incorporation, indicating D N A replication, while quiescent cells show minimal E d U incorporation. Panel B: A bar graph displays the percentage of E d U positive cells in cycling and quiescent conditions. The cycling condition shows a high percentage of E d U positive cells, while the quiescent condition shows a negligible percentage. Panel C: A histogram shows the distribution of propidium iodide (P I) staining for cycling cells and cells in quiescence for 7 days. Cycling cells exhibit a broad distribution, while quiescent cells show a narrow distribution. Panel D: A scatter plot shows C E N P-A centromere intensity levels over time as cells enter quiescence. Intensity levels decrease over time, with a significant drop at the 7-day mark. Panel E: A scatter plot shows C E N P-T centromere intensity levels in cycling and quiescent mouse 3 T 3 cells. Cycling cells exhibit higher intensity levels compared to quiescent cells. Panel H: Immunofluorescence images show cycling and 7-day quiescent mouse 3 T 3 cells stained with C E N P-C and anti-centromere (A C A) antibodies. Cycling cells show stronger staining compared to quiescent cells. Panel I: A western blot image shows cells treated with C E N P T or control s i R N A s, verifying the banding pattern for C E N P T antibody. Panel J: A western blot image shows cells treated with C E N P C or control s i R N A s, verifying the banding pattern for C E N P C antibody. Panel K: A western blot image shows cells treated with C E N P H or control s i R N A s, verifying the banding pattern for C E N P H antibody. All data is approximate.

Controls for quiescence induction and conservation of quiescent centromere behavior in mouse cells. (A) Representative images of EdU staining of cycling and quiescent cells. Cells were incubated for 48 h in EdU, a nucleotide analog that monitors DNA replication and progression through the cell cycle. Scale bar = 100 µm. (B) Graph showing the percentage of EdU-positive cells for the indicated condition. Cells were incubated for 48 h in EdU. Bars represent mean ± SD of three replicates. Mean of cycling is 95.12; mean of quiescence is 0.2. ** represents P = 0.0015, using unpaired t test with Welch’s correction. (C) Histogram showing the distribution of PI staining for cycling cells (blue) or cells in quiescence for 7 days (green) as measured by flow cytometry. (D) Graph showing CENP-A centromere intensity level over time of quiescence entry. Each point indicates the average centromere intensity level for all centromeres of a single cell, adjusted for background. Intensity values were normalized to day 0. Red line represents the median, and blue points represent average of each replicate. Points were from aggregated from six replicates. n = 440, 661, 768, 726, 686, and 555 cells for 0, 1, 2, 3, 5, and 7-day time points, respectively. * represents P < 0.05; P = 0.0297 between cycling and 7-day quiescent, averages were 1 and 0.8429, respectively. P values were calculated using unpaired t test with Welch’s correction. (E) Graph showing CENP-T centromere intensity levels in cycling and quiescent mouse 3T3 cells. Cells were quiescent for 7 days. Each point indicates the average centromere intensity level for a single cell, adjusted for background. Intensity values were normalized to cycling condition. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 206 and 223 cells for cycling and quiescent, respectively. P = 0.0031, using unpaired t test with Welch’s correction. (F) Representative immunofluorescence images of cycling and 7-day quiescent mouse 3T3 cells. Cells were stained with mouse CENP-T and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (G) Graph showing CENP-C centromere intensity levels in cycling and quiescent mouse 3T3 cells. Cells were quiescent for 7 days. Each point indicates the average centromere intensity level for a single cell, adjusted for background. Intensity values were normalized to cycling condition. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 195 and 254 cells for cycling and quiescent, respectively. P = 0.0342, using unpaired t test with Welch’s correction. (H) Representative immunofluorescence images of cycling and 7-day quiescent mouse 3T3 cells. Cells were stained with mouse CENP-C and anti-centromere (ACA) antibodies. Scale bar = 5 µm. (I) Western blot of cells treated with 50 nM CENPT or control siRNAs. Blot verifies the banding pattern for CENP-T antibody. CENP-T is the upper band. Blot was incubated in CENP-T antibody. (J) Western blot of cells treated with 50 nM CENPC or control siRNAs. Blot verifies the banding pattern for CENP-C antibody. Blot was incubated in CENP-C antibody. (K) Western blot of cells treated with 50 nM CENPH or control siRNAs. Blot verifies the banding pattern for CENP-H antibody. CENP-H is the upper band. Blot was incubated in CENP-H antibody. Source data are available for this figure: SourceData FS1.

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