Live cell imaging analysis of mitotic fidelity

Lymphoblastoid cell lines were imaged as previously described (Yost et␣al, 2017). siRNA transfections (RNAiMAX, Thermo Fisher) in HeLa ^{EGFP‐AID‐CENATAC} (40 nM siRNA) and DLD‐1 cells (50 nM siRNA) were done against CENATAC (CCDC84; Dharmacon, J‐027240‐07), GAPDH (Dharmacon, D‐001830‐01‐05), or ZRSR2 (Sigma, SASI_Hs02_00338940). In the case of CENATAC depletion in HeLa ^{EGFP‐AID‐CENATAC} cells, transfections were done in the presence of 1 mM 3‐indoleacetic acid (IAA) or ethanol (IAA vehicle) for 24 h in a 24‐well plate before the cells were re‐plated to eight‐well ibidi μ‐slides with 2 mM thymidine (for early S‐phase synchronization) and 100 μl lentivirus for CENATAC re‐expression. After 18 h, the cells were released from thymidine for 6 h and imaged in CO₂‐independent medium in a heated chamber (37°C), while air‐tight‐sealed in the well plate with parafilm. These cells were therefore imaged ˜ 48 h after siRNA‐mediated knockdown of CENATAC and ˜ 24 h after lentivirus addition. For CENATAC depletion and re‐expression in DLD‐1 cells, the lentivirus (150 μl) was immediately added together with the siRNA treatment (instead of 24 h later together with the 2 mM thymidine). These cells were therefore imaged ˜ 48 h after siRNA‐mediated knockdown of CENATAC and ˜ 48 h after lentivirus addition. For the experiments in Figs 2D and EV2A, the cells were additionally incubated with 200 nM SiR‐tubulin dye (Spirochrome) for 6 h prior to imaging to facilitate visualization of the mitotic spindle. For the depletion of ZRSR2 in both HeLa ^{EGFP‐AID‐CENATAC} and DLD‐1 cells, the cells were re‐plated to 8‐well ibidi μ‐slides with 2 mM thymidine 48 h (instead of 24 h) after transfection and therefore imaged ˜ 72 h after siRNA‐mediated knockdown of ZRSR2. Images were acquired every 3 or 5 min at 1 × 1 binning in 7 × 2.5 μm z‐stacks (RFP as in Appendix␣Figs S4 and S6 was imaged in only 1 z‐stack per position) and projected to a single layer by maximum intensity projection using NIS‐Elements Software 4.45. Imaging was performed with a Nikon Ti‐Eclipse wide‐field microscope equipped with an Andor Zyla 4.2 sCMOS Camera, 40× oil objective NA 1.3 WD 0.2 mm, and Lumencor SPECTRA X light engine. Analysis of these experiments was carried out with ImageJ software. When applicable, cells re‐expressing CENATAC variants were identified through co‐expression of cytosolic RFP (via ires‐tagRFP); RFP‐negative cells were omitted from the quantifications (Appendix␣Figs S4 and S6).