Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: ( A ) Current mitotic models of polyploidy evolution in various systems, based on existing literature (see main text). STOP signs represent cell cycle arrest or cell death. Note that polyploid cells with extra centrosomes can be arrested prior to the first mitosis (not shown) or after the first mitosis by p53-dependent pathways. ( B ) Fixed images of mSI organoids (left) and anaphase cells (middle) stably expressing EGFP-Centrin-2 (cyan) and stained with DAPI (magenta). The bar plot shows the frequencies of anaphase cells with two or more than two centrosomes in organoids of different sizes. N = 384 cells from > 300 organoids and ≥ 3 experiments. ( C ) Fixed images of mSI metaphase cells stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta). The univariate scatter plot shows metaphase DNA volume, obtained with segmentation analysis, in cells with different centrosome numbers. Colored points represent individual cells; black lines show the mean, with light and dark grey areas marking 95% confidence intervals for the mean and standard deviation, respectively. Cells are separated by pseudotime of growth, which is based on the time of fixation post seeding (48h and 96h) and the size of the organoid from which they were taken. Dashed lines indicate the minimum threshold for increased DNA volume, associated with polyploidy, defined as the mean + SD of cells with two centrosomes (see Methods). Blue shaded areas indicate cells with two centrosomes and the grey shaded area indicates cells with increased DNA content at 48h. N = 157 cells from > 100 organoids and ≥ 3 experiments. ( D ) Images show fixed bipolar mSI anaphase cells with four centrosomes clustered in a symmetric (2:2) or asymmetric (1:3) configuration. The frequency of each configuration in metaphase and anaphase is shown on the graph on the right, at pseudotime described in ( C ). N = (12, 19) cells from > 10 organoids and ≥ 3 experiments; p > 0.05. ( E ) Fixed mSI anaphase cells with more than two centrosomes exhibiting bipolar or multipolar spindles (top panel) and frequency of each type in small organoids (bottom panel). N = 27 cells from > 20 organoids and ≥ 3 experiments. ( F ) Top panel: schematic representation of the protocol for acute polyploidy induction with cytochalasin D. Numbers in yellow represent timepoints after seeding of dissociated organoids; numbers in purple time of incubation with the inhibitor and numbers in black incubation time with fresh medium before fixation (recovery time). Images on the bottom show fixed mSI anaphases after polyploidy induction. Graph shows quantification of spindle polarity types in anaphase after polyploidy induction at either 48 or 96h post seeding dissociated organoids. N = (70, 64) cells from > 60 organoids and ≥ 3 experiments. All images are maximum intensity projections. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining (tubulin channel not always shown). Statistics: Z-test for two population proportions when comparing frequencies in (B), (D) and (F); in (C) Kruskal-Wallis test with post hoc Dunn’s test. Symbols: *, P < 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. For simplicity, in (B) and (C) not all p-value symbols are shown. Cents, centrosomes; arb., arbitrary; M., middle; metaph., metaphase; anaph., anaphase; fix, fixation; inact., inactivated; multip., multipolar; ind., induced.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Standard Deviation, Incubation

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) Fixed images of mSI anaphase cells stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta). The univariate scatter plot shows the size of each organoid that contained anaphase cells with two or more than two centrosomes. N = (342, 36) cells from (> 300, > 30 organoids) and ≥ 3 experiments. (B) Fixed images of mSI metaphase cells with two and more than two centrosomes. The univariate scatter plots show metaphase plate length (N = 152, 21 cells), spindle length (N = 152, 21 cells), and cell circumference for metaphase cells (N = 152, 19 cells) with two and more than two centrosomes. Data from (> 100, > 10 organoids) and ≥ 3 experiments. (C) Fixed images of a mSI single cell in metaphase. Graph shows the frequencies of single cells with different number of centrosomes in metaphase and anaphase at 48h post seeding dissociated organoids. N = 25 cells and ≥ 3 experiments. (D) Correlation between DNA volume and cell circumference (left) or plate length (right) across metaphase cells with different centrosome numbers. Black lines show linear regression. N = 122 cells from >100 organoids and ≥ 3 experiments. (E) Images show fixed organoids and a single cell immunostained for basolateral marker E-cadherin (cyan), stained with SiR-actin (yellow) and DAPI (magenta). The second and third column show single z-planes. White arrows point to cell borders, determined by the F-actin staining. (F) Images show an example of a fixed anaphase cell after induction of polyploidy that has a distant inactivated spindle pole (arrowhead). (G) The bar plot compares the frequencies of symmetric (2:2) and asymmetric (1:3) clustering configuration of centrosomes in bipolar mSI anaphase cells with four centrosomes. Frequencies are shown for polyploidy induction at both 48h and 96h post seeding. N = (51, 43) cells from > 40 organoids and ≥ 3 experiments. All images, unless otherwise stated, are maximum intensity projections. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining. In univariate plots, colored points represent individual cells; black lines show the mean, with light and dark grey areas marking 95% confidence intervals for the mean and standard deviation, respectively. Statistics: Z-test for two population proportions when comparing frequencies in (G); Mann-Whitney U test in univariate plots in (A) and (B) and two-tailed t-test in (D). Symbols: **, P ≤ 0.01; ****, P ≤ 0.0001. Cents or cent., centrosome(s); circumfer., circumference; metaph., metaphase; anaph., anaphase; arb., arbitrary; org., organoid; ind., induced; unk. or unkn., unknown.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Single Cell, Marker, Standard Deviation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) Representative time-lapse images of a bipolar division in a mSI cell with four centrosomes stably expressing EGFP-Centrin-2 (cyan) and with leaky expression of H2B-mCherry (magenta). Plot shows distances between all tracked centrosomes in cells with four centrosomes over time. Centrosome distances that are < 6 µm apart before anaphase onset are defined as clustering (blue thin lines) and otherwise as separating (purple thin lines). N = 5 cells from > 3 organoids and ≥ 3 experiments. (B) Time-lapse images of a bipolar division in a mSI cell with two centrosomes. Plot shows centrosome distances in time (purple thin lines) for all cells with two centrosomes that had diploid-sized nuclei (see Methods). N = 23 cells from > 10 organoids and ≥ 3 experiments. In (A-B) time zero marks mitotic onset (dashed line); thick lines are mean values; shaded areas are SEM values. (C) Fixed images of mSI anaphase cells stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta). Cells are fixed 45-60 min post wash-out of 3 µM nocodazole. The bar plots show quantification of mitotic spindle polarity after nocodazole wash-out, in cells prior to (left) and during (right) anaphase. N = (131, 38) cells from (> 100, > 30) organoids and ≥ 3 experiments. (D) Fixed images of mSI anaphase cells after 30 min of treatment with 250 µM HSET inhibitor CW069. In (C-D), prior to treatment, polyploidy was induced using cytochalasin D at 48h post seeding dissociated organoids and only cells with four centrosomes were selected for analysis. The bar plots show quantification of mitotic spindle polarity in treated cells. N = 49 cells from > 40 organoids and ≥ 3 experiments. (E) Fixed images of mSI mitotic cells with two centrosomes after 3h of treatment with 30 µM Eg5 inhibitor monastrol. The bar plot shows the frequency of centrosome distances <4 µm or >4 µm, with larger cells shown separately due to possible polyploidy (see Methods). N = (81, 23) cells from (> 50, > 15) organoids and ≥ 3 experiments. Statistics: Z-test for two population proportions when comparing frequencies in (E). All images are maximum intensity projections. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining or by observing centriole pair trajectories during live-cell imaging (see Methods). The cell nuclei and chromatin groups in telophase are outlined with white dashed lines for clarity. The cell nuclei and chromatin groups in telophase are outlined with white dashed lines for clarity. NEB, nuclear envelope breakdown; BAO, before anaphase onset; noco WO, nocodazole wash-out; cents, centrosomes.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Live Cell Imaging

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) A live-imaged mature mSI organoid, 24h after seeding mechanically fragmented organoids with cells stably expressing EGFP-Centrin-2 (cyan) and with leaky expression of H2B-mCherry (magenta). (B) Representative time-lapse images of a bipolar division in a mSI cell with two centrosomes. The cell nucleus and chromatin groups in telophase are outlined with white dashed lines for clarity. (C) Plot shows centrosome distances in time (purple thin lines) for all cells with two centrosomes within the mature organoids. N = 12 cells from 2 organoids and 1 experiment. Time zero marks mitotic onset (dashed line); thick lines are mean values; shaded areas are SEM values. (D) Fixed images of mSI early and later prometaphase cells with two (first and middle row) or four centrosomes (bottom row) stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta). Cells are showing characteristics of the prometaphase spindle assembly pathway, primarily the presence of prometaphase rosettes (DAPI channel, magenta). The last column shows a simplified schematic representation of mitotic spindle (grey) and chromosome (magenta) from the images on the left. (E) The bar plot shows the fraction of prometaphase cells with two centrosomes undergoing a prometaphase spindle assembly pathway. N = 25 cells from > 20 organoids and ≥ 3 experiments. (F) The univariate scatter plot of centrosome distances at NEB (left) and mitotic time (right) across live-imaged cells from Figure (2A-B) containing two or four centrosomes grouped by nuclear size (“normal”, “large”) and centrosome clustering outcome (“clustered”, “non-clust.”) or spindle polarity in anaphase (“bipolar”, “multipolar”), respectively. N = (23, 1, 5, 2) cells from (> 10, 1, > 3, 2) organoids and ≥ 3 experiments per centrosome group (“2 cents” and “4 cents”). (G) Fixed images of mSI anaphase cells treated with 3 µM nocodazole for 2h. The univariate scatter plot shows centrosome distances in cells with four centrosomes treated with 3 µM nocodazole. Six unique distances are plotted for each cell with four centrosomes. Distances in cells with two centrosomes are shown for comparison, with large cells separated, due to possible polyploidy. N = (74, 28, 14) cells from > 50 organoids and ≥ 3 experiments in total. (H) The bar plots show the frequency of mitotic spindle polarity groups in fixed mSI anaphase cells treated with 250 µM and 400 µM HSET inhibitor CW069 for indicated durations. In (G-H), prior to treatment, polyploidy was induced using cytochalasin D at 48h post seeding dissociated organoids and only cells with four centrosomes were selected for analysis in (H). N = (80, 43) cells from (> 70, > 30) organoids and ≥ 3 experiments. All images are maximum intensity projections. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining or by observing centriole pair trajectories during live-cell imaging (see Methods). In (G) centrosome identity is assumed based solely on the centrin signal and cell boundaries. In univariate plots, colored points represent individual cells; black lines show the mean, with light and dark grey areas marking 95% confidence intervals for the mean and standard deviation, respectively. Statistics: Z-test for two population proportions when comparing frequencies in (H); in (F) ANOVA with post hoc Tukey’s HSD test; in (G) Kruskal-Wallis test with post hoc Dunn’s test. Symbols: **, P ≤ 0.01; ***, P ≤ 0.001. NEB, nuclear envelope breakdown; BAO, before anaphase onset; cents, centrosomes; Non-clust., non-clustered.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Comparison, Live Cell Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) Fixed images of mSI anaphase cells with two or more than two centrosomes, stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (not shown) and stained for F-actin with SiR-actin (not shown) and for DNA with DAPI (magenta). The bar plot shows the frequency of anaphase segregation errors, depending on centrosome number (left) or organoid size (right). Cells from large organoids are excluded from the left plot due to a low number of cells with more than two centrosomes. N = (242, 32) cells from (> 200, >30) organoids and ≥ 3 experiments. Organoid sizes reflect growth stages. Numbers of analysed cells are indicated on the data labels. Data from > 90 organoids and ≥ 3 experiments in each organoid group. (B) Frequencies of chromosome segregation errors in mSI anaphase cells with more than two centrosomes, depending on their spindle polarity. N = (20, 7) cells from (> 15, > 5) organoids and ≥ 3 experiments in each group. (C) Fixed images of mSI anaphase cells dividing within a monolayer, with two or more than two centrosomes. White rectangle marks the cell enlarged on the right. The first column shows the mSI monolayer as a three-dimensional projection. The bar plot shows the frequencies of chromosome segregation errors in mSI anaphase cells in a monolayer (“2D”) versus cells coming from organoids (“3D”) at the same timepoint after seeding dissociated organoids (48h). Numbers of analysed cells are indicated on the data labels. 3D data from > 30 organoids; ≥ 3 experiments in each group. (D) Images show fixed organoids immunostained for apicobasal polarity markers β4-integrin (cyan) and Zonula occludens-1 (ZO-1, green) and stained with SiR-actin (yellow) and DAPI (magenta). Images that show β4-integrin staining are single z-planes. (E) Fixed images of mSI anaphase cells after polyploidy induction with cytochalasin D that had separated chromatin groups lacking adjacent spindle poles, indicating chronocrisis. White arrows point to chromatin groups without adjacent poles, additionally encircled with white dashed lines. The bar plot shows the frequencies of chromosome segregation errors in anaphase cells after polyploidy induction at 48h post seeding dissociated organoids. Cells are separated in groups based on centrosome numbers and spindle polarity. Erroneous divisions coming from cells in chronocrisis are separated from other error types. Numbers of analysed cells are indicated on the data labels. Data from > 3 organoids and ≥ 3 experiments in each group. All images are maximum intensity projections, unless stated otherwise. “Uninduced” stands for native mSI organoids in which no polyploidy induction was performed, contrary to “induced polyploidy”. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining (tubulin channel not always shown). Statistics: Z-test for two population proportions in (A)-(C), (E). Symbols: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. For simplicity, in (C) not all p-value symbols are shown. Cents, centrosomes; unkn., unknown; div., division; multip. or m., multipolar; inac., inactivated.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) The univariate scatter plot shows the size of each organoid that contained anaphase cells, separated by chromosome segregation outcomes. N = (453, 50) cells from (> 400, > 40) organoids and ≥ 3 experiments per group (left). The bar plot shows the frequency of chromosome segregation errors of mSI anaphase and telophase cells in organoids of different sizes. Numbers of analysed cells are indicated on the data labels. Data from > 100 organoids and ≥ 3 experiments in each group (right). (B) The bar plot shows frequencies of each spindle polarity type in mSI metaphase cells with more than two centrosomes, in organoids and single cells. N = 8 cells from > 5 organoids and 10 single cells, both from ≥ 3 experiments. (C) The univariate scatter plot shows the DNA volume of anaphase cells that had a chromosome segregation error, separated by centrosome numbers. Shaded area (grey) marks the values of DNA volume that are above the mean + SD of cells with two centrosomes, indicating polyploidy (see Methods). N = (19, 17) cells from > 15 organoids and ≥ 3 experiments in each group. (D) Correlation between DNA volume and cell circumference (left) across anaphase cells with different centrosome numbers. Black line shows linear regression. N = 35 cells from >30 organoids and ≥ 3 experiments. The univariate scatter plot shows the DNA volume of anaphase and telophase cells that had a chromosome segregation error, separated by spindle polarity (right). Shaded area (grey) marks the values of DNA volume that are above the mean - SD of multipolar cells, indicating polyploidy (see Methods). N = (12, 6) cells from > 5 organoids and ≥ 3 experiments in each group. (E) The bar plot shows the frequencies of mSI anaphase cells with two or more than two centrosomes, depending on whether they divide in a monolayer (“2D”) or in organoids (“3D”), at the same timepoint after seeding dissociated organoids (48h). N = (119, 234) cells from > 100 organoids and ≥ 3 experiments in each group. (F) Fixed images of mSI anaphase cells stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta), dividing within a monolayer with two or more than two centrosomes. Images are maximum intensity projections. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining. In univariate plots, colored points represent individual cells; black lines show the mean, with light and dark grey areas marking 95% confidence intervals for the mean and standard deviation, respectively. Statistics: Z-test for two population proportions when comparing frequencies in (A), (B), (E); in univariate plot in (A) Kruskal-Wallis test with post hoc Dunn’s test; in (C) and (D, right): T-test for data that followed a normal distribution or Mann-Whitney U test otherwise; two-tailed t-test in (D, left). Symbols: **, P ≤ 0.01. Anaph., anaphase; org., organoids; m., middle; arb., arbitrary; cents, centrosomes; divis., division.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Standard Deviation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) Images show fixed mSI organoids immunostained for basal marker β4-integrin (cyan) and (B) apical marker Zonula occludens-1 (ZO-1, green) and stained with SiR-actin (yellow) and DAPI (magenta). Images that show β4-integrin staining are single z-planes. (C) The bar plot on the left shows the frequencies of chromosome segregation errors in mSI anaphase cells expressing EGFP-Centrin-2 (stained as in ), depending on whether they come from columnar or non-columnar organoids, examples of which are shown in (A) and (B) under “Columnar” or “Non-columnar”. The bar plot on the right shows the same data, but with additional separation of cells by pseudotime of organoid growth, which is based on the time of fixation post seeding (48h and 96h) and the size of the organoid from which they were taken. Numbers of analysed cells are indicated on the data labels. Data from > 30 organoids and ≥ 3 experiments in each group. (D) Fixed images of mSI cells stably expressing EGFP-Centrin-2 (cyan), immunostained for α-tubulin (grey) and stained for F-actin with SiR-actin (not shown) and for DNA with DAPI (magenta), after polyploidy induction with cytochalasin D exhibiting: asynchronous mitotic entry of a binuclear cell in prophase (top row); separated chromatin groups of different condensation levels and lacking adjacent spindle poles in metaphase and anaphase, indicating chronocrisis (middle and bottom row). White arrows point to a nucleus of a binuclear that is late with mitotic entry, additionally encircled with a white dashed line (top row), or chromatin groups without adjacent poles, additionally encircled with white dashed lines (middle and bottom row). (E) The bar plots show the number of anaphase cells with denoted chromosome segregation outcomes after polyploidy induction at 48h (top) or 96h (bottom) after seeding dissociated organoids. Cells are separated based on their spindle polarity and centrosome numbers (color-coded shaded areas). Data from > 30 organoids and ≥ 3 experiments. (F) The bar plot shows the frequencies of chromosome segregation errors in anaphase cells after polyploidy induction at 96h post seeding dissociated organoids, separated by centrosome numbers and spindle polarity. Erroneous divisions coming from cells in chronocrisis are separated from other error types. Numbers of analysed cells are indicated on the data labels. Data from > 3 organoids and ≥ 3 experiments in each group. (G) Fixed mSI anaphase cell with a separated chromatin group without adjacent spindle poles, indicating chronocrisis. The bar plot on the right shows the frequency of anaphase cells in chronocrisis (purple) in mSI organoids 48h after seeding dissociated organoids. N = 30 from > 20 organoids and ≥ 3 experiments. (H) The bar plot shows frequencies of spindle polarity types in anaphase cells when chronocrisis events are excluded. Cells are separated based on polyploidy induction timepoint: at 48h and 96h after seeding dissociated organoids. Only cells with four centrosomes are shown, unless the number of centrosomes is unknown. N = (54, 53) cells from > 50 organoids and ≥ 3 experiments in each group. All images are maximum intensity projections, unless stated otherwise. “Uninduced” stands for native mSI organoids in which no polyploidy induction was performed, contrary to “induced polyploidy”. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining. Statistics: Z-test for two population proportions in (A), (F), (H). Symbols: *, P < 0.05. Cents, centrosomes; unkn., unknown; no err., no error; non-col., non-columnar; col., columnar; multip., multipolar; ind., induced.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Marker, Staining, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) Representative time-lapse images of the progeny of a binuclear polyploid bipolar division in an mSI cystic organoid stably expressing H2B-mNeon (color coded by depth). Polyploidy was induced with cytochalasin D at 48h after seeding dissociated organoids. Binuclear polyploid cell is marked with a white rectangle. Time zero is mitotic onset. Numbers in left corners denote cell identity with respect to time and mother cell of origin. Nuclei of a binuclear cell and chromatin groups in anaphases are outlined with white dashed lines for clarity. Images of anaphase are smoothed with 0.5 sigma Gaussian blur. (B) The bar plot shows the frequencies of anaphase spindle polarity types in the first and second division after polyploidization, in live- imaged mSI cells expressing H2B-mNeon. N = (47, 45) cells from > 3 organoids and ≥ 3 experiments for each group. (C) The bar plot shows the number of the daughter cells undergoing denoted proliferation outcomes, based on their mother (first division) polarity status and presence of segregation errors during anaphase. Non-proliferative outcome is defined as daughter cells not dividing or dying for >30h after the mother division (see Methods). N = 72 cells from 3 organoids and 3 experiments. (D) The bar plot shows the frequencies of daughter cell proliferation outcomes depending on the segregation error status of the mother cell after polyploidy induction and in DMSO-treated controls. Legend as in (C). Number of cells is denoted on the data labels. Data from 3 organoids and 3 experiments. (E) The bar plot shows the frequency of multipolar divisions that resulted in either 2 or 3 daughter cells. Only first multipolar divisions after polyploidization were analysed. N = 11 cells from > 3 organoids and ≥ 3 experiments in total. (F) The bar plot shows the number of granddaughter cells undergoing denoted proliferation outcomes, separated based on spindle polarity type and chromosome segregation error outcome of the first (mother) division. All shown granddaughters come from bipolar daughters. N = 31 cells from 2 organoids and 2 experiments. (G) The univariate scatter plot of mitotic time for indicated spindle polarity types, shown separately for DMSO-treated control cells and cells with induced polyploidy. Shaded area (grey) separates cells in their first division after polyploidization from those in second division (right white area) and DMSO-treated control cells (left white area). N = (93, 34, 11, 30, 8, 2) cells from > 3 organoids and ≥ 3 experiments in total. (H) The univariate scatter plot of cycle duration for indicated generations. Cycle times of binucleated daughters are separated (“1 st bi.”). Data is shown separately for DMSO-treated control cells and cells with induced polyploidy. Shaded area (grey) separates cells in their first generation after polyploidization (daughter) from those in second generation (granddaughter, right white area) and DMSO-treated control cells (left white area). N = (37, 37, 8, 18) cells from > 3 organoids and ≥ 3 experiments in total. (I) Large-field images of fixed mSI organoids stably expressing EGFP-Centrin-2 (not shown), stained for F-actin with SiR-actin (red) and for DNA with DAPI (blue). Organoids were either treated with DMSO for control (left) or cytochalasin D to induce polyploidy (right), 48h after seeding dissociated organoids. Time of fixation is 48h after DMSO or inhibitor wash-out. Images in the right upper corners are enlargements of representative organoids marked with white rectangles. (J) Quantification of organoid types (see Methods) from (I). N = (530, 424) organoids from ≥ 3 experiments in total. (K) Fixed images of human non-cancer colonic organoids, immunostained for α-tubulin (grey) and centrin-3 (cyan), stained for F-actin with SiR-actin (yellow) and for DNA with DAPI (magenta). Single z-planes of a mitotic cell marked with a white rectangle are enlarged on the right to show centrosomes. White circles mark centriole pairs for which centrosome identity was confirmed using α-tubulin staining. (L) Fixed images of human non-cancer colonic organoids, stained as in (K), with single z-planes of a binuclear cell marked with a white rectangle enlarged on the right. Nuclei of a binuclear cell are outlined with white dashed lines. All images are maximum intensity projections, unless otherwise stated. In univariate plots, colored points represent individual cells; black lines show the mean, with light and dark grey areas marking 95% confidence intervals for the mean and standard deviation, respectively. Statistics: Z-test for two population proportions when comparing frequencies in (B), (D) and (J); in univariate plots: Kruskal-Wallis test with post hoc Dunn’s test. Symbols: *, P < 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. For simplicity, in (D) not all p-value symbols are shown. Multi or M, multipolar; non-prolif., non-proliferating; unkn., unknown; polar., polarity; gen., generation; B, bipolar; BB, binuclear bipolar; bi., binuclear; polyp., polyploidy; org., organoids.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Control, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

doi: 10.64898/2026.03.25.714196

Figure Lengend Snippet: (A) The bar plot shows the frequencies of chromosome segregation errors in the first division of live-imaged mSI cells after polyploidy induction with cytochalasin D at 48h after seeding dissociated organoids. Cells are further separated based on spindle polarity. N = (33, 11) cells from > 3 organoids and ≥ 3 experiments. Representative live-imaged polyploid multipolar (top) and bipolar (bottom) erroneous anaphase of mSI cells stably expressing H2B-mNeon (color coded by depth and smoothed with 0.5 sigma Gaussian blur). (B) Large-field images of fixed mSI organoids stably expressing EGFP-Centrin-2 (not shown), stained for F-actin with SiR-actin (red) and for DNA with DAPI (blue). Organoids were either treated with DMSO for control (top) or cytochalasin D to induce polyploidy (bottom), 48h after seeding dissociated organoids. Time of fixation is 48h after DMSO or inhibitor wash-out. Images in corners are enlargements of representative organoid groups marked with white rectangles. All images are maximum intensity projections. Statistics: Z-test for two population proportions in (A). Symbols: **, P ≤ 0.01.

Article Snippet: In the organoid formation assay experiment, samples were fixed with 4% paraformaldehyde, stained overnight with 1 μM SiR-actin (Spirochrome) and shortly with DAPI (1 μg/mL).

Techniques: Stable Transfection, Expressing, Staining, Control