Journal: bioRxiv

Article Title: Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes

doi: 10.1101/2022.11.08.515699

Figure Lengend Snippet: (a) Immunofluorescence staining of control and acentriolar RPE-1 TP53-/- or U2OS cells. Acentriolar cells were created by treatment with 125 nM centrinone (CNONE) or by deletion of SASS6 . DAPI is shown in blue, γ-tubulin in green, and centrin-3 in magenta. Insets are shows for cells in which centrosomes were present. (a’) Quantification of a. Graphed are means and S.E.M. Significance was determined through a Fisher’s exact test. n =100 cells per condition. (b) Live phase contrast imaging showing example mitosis in U2OS cells pre-treated with DMSO or centrinone (CNONE) for 10 days. Green bars below cells indicate the duration of nuclear envelope breakdown (NEBD) through the onset of anaphase, while magenta bars below cells indicate the duration of the completion of anaphase, telophase, and cytokinesis. (b’) Mitotic durations from NEBD through metaphase (green) and anaphase, telophase, and cytokinesis (magenta) as in (b). Time is in minutes. Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t -test. n 20 cells per condition. (c) Live phase contrast imaging showing example mitosis in U2OS cells pre-treated with DMSO or centrinone (CNONE) for 10 days before being imaging in 1 μM CFI-402257. (c’) Quantification of mitotic duration in cells of indicated genotype or treatment in DMSO (grey) or CFI-402257 (blue). Time is in minutes. Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t -test. n 25 cells per condition. (d) Quantification of daughter cell fate of cells of the given pre-treatment imaged in DMSO or CFI-402257 (1 μM). Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. (e) Confocal timelapse imaging of U2OS cells pretreated for 10 d with 125 nm centrinone and imaged in 1 μM CFI-402257. Shown are endogenously tagged α-tubulin ( GFP-TUBA1B ) and DNA (Sir-Hoechst). (f) Quantification of daughter cells fates of cells of the given pre-treatment imaged in DMSO or CFI-402257 (1 μM) with concurrent treatment with proTAME (12 μM for RPE1 cells, 24 uM for U2OS cells). Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. n 40 cells per condition. In all cases, not significant (n.s.) denotes p >0.05, * denotes p<0.05, **denotes p<0.01, and *** denotes p<0.001. All scale bars: 10 μm.

Article Snippet: Primary antibodies used for immunofluorescence (IF) and Western blot (WB): mouse IgG1 anti- α -tubulin, clone DM1A (1:1,000IF, 1:5,000WB, Sigma-Aldrich); mouse IgG2 b anti-centrin3, clone 3e6 (1:1,000 IF, Novus Biological); mouse IgG1anti- γ -tubulin, clone GTU-88 (1:1000 IF, Sigma-Aldrich); rabbit anti-CP110, 12780-1-AP (1:200 IF, Proteintech), mouse IgG2 b anti-Lys-40-acetylated α -tubulin, clone 6-11B-1 (1:1,000 IF, 1:10,000 WB, Sigma-Aldrich; mouse IgG2 b anti- β -tubulin, clone D3U1W (1:500 IF, Cell signaling Technology); mouse IgG1 anti-Cas9, clone 7A9 (1:1000 WB, BioLegend).

Techniques: Immunofluorescence, Staining, Control, Imaging

Journal: bioRxiv

Article Title: Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes

doi: 10.1101/2022.11.08.515699

Figure Lengend Snippet: (a) Confocal timelapse imaging of U2OS cells pretreated for 10d with either DMSO or centrinone (125 nM). Shown are endogenously tagged α-tubulin ( GFP-TUBA1B ) and DNA (Sir-Hoechst). Time indicates minutes before (-) or after (+) NEBD. (a’) Proportion of cells with 2 foci of microtubules before NEBD based on experiments as in (a). (b) Live phase imaging of RPE1 TP53 -/- or U2OS cells imaged in 1μM CFI-402257 or 5 μM STLC and 1μM CFI-402257 together. (b’) Quantification of mitotic outcome of RPE1 TP53 -/- or U2OS cells imaged in DMSO, 1μM CFI-402257, 5 μM STLC, or 5 μM STLC and 1μM CFI-402257 together. Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. n =50 cells per condition (c) Confocal timelapse imaging of U2OS cells imaged in 5μM STLC with 1μM CFI-402257. Shown are endogenously tagged α-tubulin ( GFP-TUBA1B ) and DNA (Sir-Hoechst). Asterisk indicated extruded chromosomes. Arrow indicated midbody-like α-tubulin structure. (d) Graphical summary of results. In all cases, not significant (n.s.) denotes p >0.05 and *** denotes p<0.001. All scale bars: 10 μm.

Article Snippet: Primary antibodies used for immunofluorescence (IF) and Western blot (WB): mouse IgG1 anti- α -tubulin, clone DM1A (1:1,000IF, 1:5,000WB, Sigma-Aldrich); mouse IgG2 b anti-centrin3, clone 3e6 (1:1,000 IF, Novus Biological); mouse IgG1anti- γ -tubulin, clone GTU-88 (1:1000 IF, Sigma-Aldrich); rabbit anti-CP110, 12780-1-AP (1:200 IF, Proteintech), mouse IgG2 b anti-Lys-40-acetylated α -tubulin, clone 6-11B-1 (1:1,000 IF, 1:10,000 WB, Sigma-Aldrich; mouse IgG2 b anti- β -tubulin, clone D3U1W (1:500 IF, Cell signaling Technology); mouse IgG1 anti-Cas9, clone 7A9 (1:1000 WB, BioLegend).

Techniques: Imaging

Journal: bioRxiv

Article Title: Post-mitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells

doi: 10.1101/2022.05.25.493455

Figure Lengend Snippet: Increase in centriole number and centrosome number during TGC differentiation A. Confocal microscopy images of tissue sections of a mouse conceptus (e9.5) transgenic for eGFP-centrin2/Arl13b-mCherry . Sections correspond to the TGC layer described in 1A. DAPI (cyan) was used to visualize nuclei, and the native eGFP-centrin2 fluorescence to visualize centrioles (white). TGCs were identified by nuclear size relative to adjoining cells. Scale bars: overview, 10 µm; inset 2µm. B. Microscopic images of differentiating TGCs at t=0d, t=6d, and t=10d. Cells were fixed and labeled with antibodies to mark the centrosome ( γ -tubulin, yellow) and centrioles (centrin, magenta). DAPI (cyan) was used to visualize nuclei. Centrioles from cells indicated with dashed lines are shown at higher magnification in insets. The t=10d images are from a large TGC whose boundary is beyond shown field of view. Scale bars: overview, 10 µm; inset 2µm. C. Quantification of centriole number throughout TGC differentiation as measured by centrin immunofluorescence, shows an increase in centriole number as cells differentiate. The percent of cells with the indicated centriole numbers was calculated in three independent experiments. For each experiment, a minimum of 60 cells per condition were counted; bars represent the mean percent of cells. Error bars represent the standard error of the mean (SEM). D. Quantification of centrosome number throughout TGC differentiation time course, as marked by γ -tubulin immunofluorescence. The percent of cells with the indicated centriole numbers was calculated in three independent experiments. For each experiment, a minimum of 60 cells per condition were counted; bars represent the mean percent of cells. Error bars represent the standard error of the mean (SEM). E. Correlation of centriole and centrosome number per cell as identified by centrin and γ -tubulin immunofluorescence, respectively, at the beginning of differentiation (t=0d) middle of differentiation (t=6d) and end of differentiation (t=10d) from the data in C and D, shading opacity of each point is 10%.

Article Snippet: Primary antibodies used were mouse monoclonal anti- γ -tubulin (Sigma-Aldrich, clone GTU88, IgG1; used at 1:5,000 dilution in PBS-BT), mouse monoclonal anti-centrin3 (Novus Biologicals, clone 93E6, IgG2b; 1:2,000), mouse monoclonal anti-α-tubulin (Abcam, clone DM1α, IgG1; 1:4,000, St. Louis, MO), mouse monoclonal anti-centrin (Sigma-Aldrich, clone 20H5, IgG2a; 1:200), mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, clone 6-11B-1, 1:1000) rabbit polyclonal anti-POC5 (A303-341A-T, Bethyl Laboratories, 1:500), rabbit polyclonal anti-CP110 (12780-1-AP, Proteintech, 1:200), rabbit polyclonal anti-CNAP1/Cep250 (Proteintech, 14498-1-AP, 1:500), rabbit polyclonal anti-Pericentrin (Abcam, ab4448, 1:500), rabbit polyclonal anti-CDK5RAP2 (Sigma-Aldrich, 06-1398, 1:1000), and rabbit polyclonal anti-Cep164 (purified from serum of immunized animals as described in , 1:1,000).

Techniques: Confocal Microscopy, Transgenic Assay, Fluorescence, Labeling, Immunofluorescence

Journal: bioRxiv

Article Title: Post-mitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells

doi: 10.1101/2022.05.25.493455

Figure Lengend Snippet: Amplified centrioles in TGCs acquire microtubule nucleation competence A. Expansion microscopy images of TGCs demonstrating supernumerary centriole content with expected morphology. Expanded TGCs were stained with antibodies to mark the centrioles (acetylated tubulin) and PCM ( γ -tubulin) and were counterstained with DAPI, to mark nuclei. Scale bars = 10 µm, inset = 2 µm (expansion). B. Immunofluorescence images showing localization of various centrosome proteins in TGCs with amplified centrosomes. Cells were fixed and labeled with antibodies to label structural, PCM and appendage proteins as indicated above each panel. Scale bar = 10 µm, inset = 1 µm C. Immunofluorescence images showing TSCs (top) and TGCs (bottom) in a microtubule regrowth experiment. First panel shows cells just prior to washout (0 Min). Second and third panels show cells shortly after washout (5 min and 15 min). Cells were fixed and labeled with antibodies to mark microtubules (α-tubulin, yellow), centrioles (centrin, magenta) and nuclei (DAPI, cyan). A-C Scale bars = 10 µm, insets in C are shown at 7x magnification.

Article Snippet: Primary antibodies used were mouse monoclonal anti- γ -tubulin (Sigma-Aldrich, clone GTU88, IgG1; used at 1:5,000 dilution in PBS-BT), mouse monoclonal anti-centrin3 (Novus Biologicals, clone 93E6, IgG2b; 1:2,000), mouse monoclonal anti-α-tubulin (Abcam, clone DM1α, IgG1; 1:4,000, St. Louis, MO), mouse monoclonal anti-centrin (Sigma-Aldrich, clone 20H5, IgG2a; 1:200), mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, clone 6-11B-1, 1:1000) rabbit polyclonal anti-POC5 (A303-341A-T, Bethyl Laboratories, 1:500), rabbit polyclonal anti-CP110 (12780-1-AP, Proteintech, 1:200), rabbit polyclonal anti-CNAP1/Cep250 (Proteintech, 14498-1-AP, 1:500), rabbit polyclonal anti-Pericentrin (Abcam, ab4448, 1:500), rabbit polyclonal anti-CDK5RAP2 (Sigma-Aldrich, 06-1398, 1:1000), and rabbit polyclonal anti-Cep164 (purified from serum of immunized animals as described in , 1:1,000).

Techniques: Amplification, Microscopy, Staining, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Post-mitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells

doi: 10.1101/2022.05.25.493455

Figure Lengend Snippet: Model of Centriole and Centrosome Amplification in Endocycling Murine TGCs Cartoon diagram showing (left) a TSC with two centrosomes (yellow dots) with either two or four centrioles (magenta barrels) and ratios of γ -tubulin (to identify centrosomes) and centrin (to identify centrioles) given below. (Right) TGCs during differentiation with a variable number of centrosomes (yellow dots) and centrioles (magenta barrels). Most TGCs disengage their paired centrioles to reach a centrin to γ -tubulin ratio of 1:1. Some TGCs undergo additional centriole/centrosome amplification that also ultimately resolves to a 1:1 ratio of centrin to γ -tubulin at the end of the endocycle. Graphic was created with Biorender.com.

Article Snippet: Primary antibodies used were mouse monoclonal anti- γ -tubulin (Sigma-Aldrich, clone GTU88, IgG1; used at 1:5,000 dilution in PBS-BT), mouse monoclonal anti-centrin3 (Novus Biologicals, clone 93E6, IgG2b; 1:2,000), mouse monoclonal anti-α-tubulin (Abcam, clone DM1α, IgG1; 1:4,000, St. Louis, MO), mouse monoclonal anti-centrin (Sigma-Aldrich, clone 20H5, IgG2a; 1:200), mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, clone 6-11B-1, 1:1000) rabbit polyclonal anti-POC5 (A303-341A-T, Bethyl Laboratories, 1:500), rabbit polyclonal anti-CP110 (12780-1-AP, Proteintech, 1:200), rabbit polyclonal anti-CNAP1/Cep250 (Proteintech, 14498-1-AP, 1:500), rabbit polyclonal anti-Pericentrin (Abcam, ab4448, 1:500), rabbit polyclonal anti-CDK5RAP2 (Sigma-Aldrich, 06-1398, 1:1000), and rabbit polyclonal anti-Cep164 (purified from serum of immunized animals as described in , 1:1,000).

Techniques: Amplification