GAS2L1 and its Ser352 phosphorylation are required for proper spindle organization and chromosome segregation. (A) RPE-1 parental cells and sublines were stained for microtubules (anti-α-tubulin) and centrosomes (anti-γ-tubulin). The sublines examined were gas2l1-knockout line (gas2l1 KO) and the knockout line stably expressing GFP-GAS2L1 (WT) or mutants (S352A and S352D). Angles between the spindle pole axis and the metaphase plate axis were determined from three repeats and are presented in a dot plot: n = 143 (parental), 123 (gas2l1 KO), 131 (WT), 135 (S352A), and 136 (S352D). Midline: mean; upper and low lines: SD. One-way ANOVA; **, P < 0.001; n.s., not significant. (B) Time-lapse imaging of RPE-1 parental cells and sublines after incubation with 0.1 µg/ml Hoechst 33342. Incidence of chromosome-segregation defects (bridging and lagging chromosomes) was quantified from three independent experiments: n = 113 (parental), 151 (gas2l1 KO), 125 (WT), 118 (S352A), and 124 (S352D). Data are presented as means ± SD of three experimental repeats. χ² test; *, P < 0.01; **, P < 0.001; n.s., not significant. Scale bars, 5 µm. (C) Times of centrosome disjunction occurrence were determined from cells exhibiting normal segregation or mis-segregation of chromosomes. Data are presented as means ± SEM. One-way ANOVA; **, P < 0.001.