GAS2L1 and its Ser352 phosphorylation regulate centrosome separation and facilitate mitotic progression. (A) Representative time-lapse sequences of parental RPE-1 cells and the indicated sublines before and during mitosis. Images are labeled with times relative to NEBD; t = 0, time of NEBD; onset times of centrosome disjunction are shown in yellow. Boxed areas are enlarged. Arrowheads: centrosomes; arrows in gas2l1^−/− cells and S352A-expressing gas2l1^−/− cells: chromosome bridges. Scale bar, 5 µm. (B) Intercentrosomal distances measured before NEBD from cells in A; t = 0, time of NEBD. (C) Dot plot of times of centrosome disjunction (d > 2 µm) occurrence. Each dot represents a single cell with data collected. Midline: mean; upper and lower lines: SD. (D) Durations of mitosis and different mitotic stages presented as means ± SEM. (C and D) Data were quantified from three different experiments: n = 122 (parental), 107 (gas2l1 KO), 118 (WT), 96 (S352A), and 121 (S352D). One-way ANOVA; *, P < 0.01; **, P < 0.001; ***, P < 0.0001; n.s., not significant.