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