Journal: bioRxiv

Article Title: A motor-based approach to induce chromosome-specific mis-segregations in human cells

doi: 10.1101/2022.04.19.488790

Figure Lengend Snippet: A . IF for pericentrin, GFP, H3S10ph of U-2 OS TetO cells in metaphase. The TetO locus (GFP focus) is aligned on the metaphase plate (TetO in) in control condition (rTetR-GFP), and co-localizing with one of the two pericentrin-marked centrosomes (TetO out) in rTetR-GFP-Kin14VIb-expressing cells. B, C . IF for CENP-C, GFP, H3S10ph of U-2 OS TetO cells in metaphase and expressing rTetR-GFP-Kin14VIb. B . Quantification of the fraction of cells with the indicated orientation of the duplicated TetO locus or TetO chromosome. Experiment was performed in duplicate (mean ± SD, n ≥ 29 per condition, per experiment). *** p<0.001 (Fisher’s exact test, comparing the fraction of cells with the TetO locus in vs. out in ctrl vs. Kin14VIb expressing cells). C . Representative images of the different orientations of the TetO chromosome and the sister KTs are shown. DNA was visualized by DAPI. Scale bar = 5 µm. Numbers below the images depict the frequency of the indicated orientation of the sister-KTs of the duplicated TetO chromosome with its p arm sticking out towards the spindle pole (arms out). KT orthogonal: inter-sister KT axis (dotted line) perpendicular to metaphase plate; KT parallel: inter-sister KT axis is (near-)parallel the metaphase plate; KT aligned: sister KTs buried inside the metaphase plate. D . Quantification of Mad1 status of KTs nearby the TetO locus. Representative IF images are shown in Figure S1. In case the KTs of the TetO chromosome were aligned, we scored whether KTs in the vicinity of the TetO locus were Mad1+. In case the KTs of the TetO chromosome could be distinguished in rTetR-GFP-Kin14VIb-expressing cells (with either orthogonal or parallel oriented KTs), we scored if at least one of the KTs was Mad1+. Experiments were performed in duplicate (mean ± SD, n ≥ 27 per condition, per experiment). Ns = not significant (χ test, comparing the fraction of cells with the Mad1-vs. Mad1+ in ctrl cells vs. Kin14VIb cells with aligned KTs, or ctrl cells vs. Kin14VIb cells with orthogonal/parallel KTs).

Article Snippet: The following primary antibodies were used: GFP booster (Chromotek, gba488), mouse anti-yH2AX (Millipore 05-636), mouse anti-H3S10ph (Millipore, 05-806), rabbit anti-H3S10ph (Millipore, 06-570), guinea pig anti-CENP-C (MBL, PD-030), mouse anti-Mad1 (Millipore, MABE867), rabbit anti-pericentrin (Abcam, ab4448), mouse anti-Cas9 (Diagenode, C15200203), rabbit anti-RFP (Rockland, ROCK600-401-379).

Techniques: Expressing

Journal: bioRxiv

Article Title: A motor-based approach to induce chromosome-specific mis-segregations in human cells

doi: 10.1101/2022.04.19.488790

Figure Lengend Snippet: A, D . IF for CENP-C, GFP, H3S10ph of U-2 OS TetO cells in anaphase (A) and telophase (B) expressing either rTetR-GFP (ctrl) or rTetR-GFP-Kin14VIb. Representative images of the most frequently observed anaphase and telophase errors in cells with either a 1-1 (majority of events in ctrl), or 2-0 (majority of events in Kin14VIb) TetO locus distribution. DNA was visualized with DAPI. Scale bar = 5 µm. White dashed line depicts the pole-pole mid-axis, while the red dashed line indicates the TetO-TetO axis (A, 1 st row). B, E . Frequencies of the different types of segregation errors observed during anaphase (B) and telophase (E). To define anaphase and telophase segregation errors involving the TetO chromosome, errors along the TetO-TetO axis or pole-pole mid-axis (collectively called: (mid-)axis), were distinguished from errors that were observed to occur not along one of these axis (bridge/lagger elsewhere). Experiments were performed in duplicate (mean ± SD, n ≥ 23 per condition, per experiment). *** p<0.001 (Fisher’s exact test, comparing the fraction of cells displaying no error vs. 1 or 2 arms stretched in ctrl vs. Kin14VIb cells in anaphase (B), or cells displaying no error vs. bridge/lagger/spike on the (mid-)axis in ctrl vs. Kin14VIb cells in telophase (E)). C . Schematic representation of the most frequently observed consequence of Kin14VIb binding to the subtelomeric TetO locus of chromosome 1 in U-2 OS-TetO cells during metaphase and anaphase.

Article Snippet: The following primary antibodies were used: GFP booster (Chromotek, gba488), mouse anti-yH2AX (Millipore 05-636), mouse anti-H3S10ph (Millipore, 05-806), rabbit anti-H3S10ph (Millipore, 06-570), guinea pig anti-CENP-C (MBL, PD-030), mouse anti-Mad1 (Millipore, MABE867), rabbit anti-pericentrin (Abcam, ab4448), mouse anti-Cas9 (Diagenode, C15200203), rabbit anti-RFP (Rockland, ROCK600-401-379).

Techniques: Expressing, Binding Assay

Journal: eLife

Article Title: Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

doi: 10.7554/eLife.27574

Figure Lengend Snippet:

Article Snippet: Antibody , mouse anti-H3S10ph , Cell Signaling Technology , RRID: AB_331748 , .

Techniques: Expressing, Construct