Centrosome number, DNA content, and shape influence mitotic spindle assembly. (A and B) Images of diploid (A) and polyploid (B) NBs labeled with Plp (green and gray) and Cnn (red and gray) antibodies. DNA in blue. (C and D) Stills of time-lapse videos of mitotic diploid (C) and polyploid (D) NBs expressing Histone 2B-RFP (red) and Spd2-GFP (green and gray in the bottom insets), corresponding to DNA and centrosomes. Time of mitosis is represented in minutes:seconds. Time 00:00 corresponds to NEBD. Schematic representations of mitosis above the stills. (A–D) Orange circles highlight NBs. (E, G, and J) Representative images of Cytosim-based simulations of spindle assembly around the DNA represented as mobile beads (E and G) or immobile plate (J). DNA is in red; MTs, green lines; and centrosomes, green circles. (E) Simulations with fixed DNA content and variable number of centrosomes. (F) Graph bars showing the percentage of simulations in each category (n = 30 simulations/condition). (G) Simulations with fixed number of 30 centrosomes and increasing number of mobile DNA beads. (H and I) Dot plots representing the number of DNA beads (H) and DNA shape aspect ratio (I) in simulations with bipolar or multipolar status (n = 45 simulations). Statistical significance was determined using a Mann-Whitney test. Error bars represent the mean ± SD and p the P value. (J) Simulations with fixed number of 30 centrosomes and different DNA shape configurations. (K) Graph bars of the percentage of simulations in each category (n = 20 simulations/condition).