For labeling with EdU, H2B-eGFP Tcf3−/− ESCs were pulsed with EdU (0.5 μM; Invitrogen) for two consecutive periods of 12 hours [according to Elabd et al. (43)] before cell fusion with H2B-mRFP NPCs. Cell hybrids were sorted immediately after fusion and were directly plated into an uncoated μ-slide eight-well grid-500 coverslip (Ibidi). After attachment, cells were imaged for 15 to 16 hours using the Andor Revolution XD confocal microscope. After the time-lapse, cells were washed with PBS and were fixed and permeabilized at room temperature with 4% PFA for 10 min and 0.3% Triton X-100 for 15 min. Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click chemistry”) for EdU labeling was performed using Alexa Fluor 647 Azide, Triethylammonium Salt (Thermo Fisher Scientific, A10277). The grid on the wells was used to localize the imaged cells after fixation. Images were taken with a Leica TCS SP5 AOBS (inverted microscope) and were analyzed and processed using Fiji (ImageJ).

To calculate the probability that a 4n cell (containing 40 ESC and 40 NPC chromosomes) segregates the parental chromosomes in a nonrandom fashion in tripolar mitosis, we considered (i) that each chromosome segregates independently of the others and of its parental origin and (ii) that the two chromatids of the same chromosome cannot segregate to the same daughter cell. The probability of a nonrandom segregation under these conditions is 1/1.84 × 1023.

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