Figure 4.
Figure 4. Arpp19 ablation induces dramatic mitotic phenotypes. (A) Arpp19Lox/Lox MEFs that express H2B-mCherry were transduced with GFP or GFP-Cre adenoviruses and kept in starvation conditions. After 3 d, cells were forced to reenter the cell cycle. Cell morphology and chromosomes were monitored by live microscopy every 5 min using transmitted light and 561-nm LED, respectively. The percentage of cells entering mitosis (based on cell rounding and chromosome condensation) over time was quantified by analyzing time-lapse images. (B) Mitosis duration (in minutes; mean ± SEM of four biological replicates) was calculated as the time from cell rounding and onset of chromosome condensation to chromosome segregation. (C) Mitotic progression was monitored in cells treated as in A, using live spinning-disk confocal microscopy. Representative still images of the different phenotypes observed in Videos 1, 2, 3, 4, and 5. Time after prometaphase onset is indicated. (D) Representative confocal sections of Arpp19Lox/Lox MEFs transduced with GFP or GFP-Cre adenoviruses. Bars, 10 µm. Cells exhibiting the indicated phenotypes were counted and are represented in a bar graph. (E) Cells in D were used for immunocytochemistry with anti-CREST and anti-BUBR1 antibodies and DAPI staining. Images were acquired by confocal microscopy. Spots corresponding to BUBR1 expression were automatically detected with Imaris in 3D images and manually corrected using CREST staining. The mean BUBR1 signal intensities were exported to Excel files for further analysis. The histogram shows the quantification of BUBR1 signal intensities (mean ± SD from 1,359 and 1,750 kinetochores from two biological replicates). Bars, 5 µm. Bar magnifications, 1 µm. (F) The spinning disk confocal movies from three different experiments were analyzed to quantify the number of cells displaying normal mitosis or the indicated phenotypes and are represented as the percentage of total cells (GFP or GFP-Cre). (G) Cells in C that underwent decondensation in prometaphase or after anaphase onset were counted and are represented. (H) GFP-Cre–expressing cells showing normally condensed or decondensed chromosomes were classified according to the indicated mitotic phenotypes.

Arpp19 ablation induces dramatic mitotic phenotypes. (A) Arpp19 Lox/Lox MEFs that express H2B-mCherry were transduced with GFP or GFP-Cre adenoviruses and kept in starvation conditions. After 3 d, cells were forced to reenter the cell cycle. Cell morphology and chromosomes were monitored by live microscopy every 5 min using transmitted light and 561-nm LED, respectively. The percentage of cells entering mitosis (based on cell rounding and chromosome condensation) over time was quantified by analyzing time-lapse images. (B) Mitosis duration (in minutes; mean ± SEM of four biological replicates) was calculated as the time from cell rounding and onset of chromosome condensation to chromosome segregation. (C) Mitotic progression was monitored in cells treated as in A, using live spinning-disk confocal microscopy. Representative still images of the different phenotypes observed in Videos 1, 2, 3, 4, and 5. Time after prometaphase onset is indicated. (D) Representative confocal sections of Arpp19Lox/Lox MEFs transduced with GFP or GFP-Cre adenoviruses. Bars, 10 µm. Cells exhibiting the indicated phenotypes were counted and are represented in a bar graph. (E) Cells in D were used for immunocytochemistry with anti-CREST and anti-BUBR1 antibodies and DAPI staining. Images were acquired by confocal microscopy. Spots corresponding to BUBR1 expression were automatically detected with Imaris in 3D images and manually corrected using CREST staining. The mean BUBR1 signal intensities were exported to Excel files for further analysis. The histogram shows the quantification of BUBR1 signal intensities (mean ± SD from 1,359 and 1,750 kinetochores from two biological replicates). Bars, 5 µm. Bar magnifications, 1 µm. (F) The spinning disk confocal movies from three different experiments were analyzed to quantify the number of cells displaying normal mitosis or the indicated phenotypes and are represented as the percentage of total cells (GFP or GFP-Cre). (G) Cells in C that underwent decondensation in prometaphase or after anaphase onset were counted and are represented. (H) GFP-Cre–expressing cells showing normally condensed or decondensed chromosomes were classified according to the indicated mitotic phenotypes.

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