Fig. 4

PRPF8 depletion impedes mitotic progression. a PRPF8 depletion in Cal51 cells results in a fivefold increase in the percentage of mitotic cells compared with control siRNA-treated cells. Plots show the mean from three independent experiments ± standard error of the mean. Statistically significant pairwise comparison is shown (***p < 0.001). b U2OS cells stably expressing green fluorescent protein (GFP)-tagged histone H2B, depleted of PRPF8, and monitored by phase-contrast time-lapse microscopy spend >120 min in mitosis, as opposed to control siRNA-treated cells, in which mitosis lasts, on average, <60 min. The bar graph depicts time spent in mitosis, measured from nuclear envelope breakdown to anaphase. c, d Live-cell imaging of U2OS GFP-H2B cells depleted of PRPF8 reveals multiple mitotic abnormalities, including unaligned chromosomes, anaphase bridges and multipolar divisions, indicated by arrows and arrowheads. Severe defects in both chromosome alignment and segregation are also observed in some cells. Representative images are shown in (d); defects are quantified in (c). e Introduction of properly spliced and processed mRNA into PRPF8-depleted cells suffices to partially rescue the observed mitotic defects. Control siRNA-treated and PRPF8-depleted cells were transfected with properly spliced and processed mRNA and analyzed for mitotic index. Plots represent the mean from three independent experiments ± standard error of the mean. Statistically significant pairwise comparisons are shown (**p < 0.01, ***p < 0.001)