The treatment of a stable AID-EWSR1/AID-EWSR1 DLD-1 cell line with Auxin induces an efficient degradation of AID-EWS. (A). Schematic diagram of OsTIR1 and AID-EWSR1 DNA construct. OsTIR1: plant driven E3 ligase, Bla ^r : Blasticidine resistance gene, Hyg ^r : Hygromycin resistance gene, mAID: mini Auxin-Inducible Degron tag, 3XFLAG; three tandem repeat of FLAG tag. The OsTIR1 construct was inserted at the RCC1 locus, and mAID construct was inserted to both EWSR1 loci (homozygous). (B). Representative images of western blotting using anti-FLAG (top panel), anti-EWSR1 (middle panel) and anti-β actin (bottom panel) obtained from AUX- and AUX + cells in the presence of Auxin at given time point (15 h, 18 h and 25 h). (C). Relative intensity of the bands of western blotting visualized with anti-EWSR1 (normalized to the bands with anti-β-actin) obtained from AUX- and AUX + cells (n = 3 experiments). Graph shows the mean of each group with Standard Deviation (SD). (n = 3 experiments). ^**** p < 0.0001 (Two tailed paired t-test). (D). Representative images of immunocytochemistry using anti-FLAG (green) and DAPI (blue) obtained from AUX- and AUX + cells. Scale bar = 10 um.

The EWSR1 knockdown promotes induction of lagging chromosome during anaphase. (A). Schematic diagram for the mitotic synchronization of EWSR1 knockdown cells using Thymidine and Nocodazole, followed by the mitotic shake-off and release from the mitotic arrest for 60 min. (B). Representative images of chromosomes; normal (top panel), lagging chromosome (middle panel) and chromosome bridge (bottom panel). Yellow square highlights the magnified images of either lagging chromosome or chromosome bridge. Chr.; chromosome, Scale bar = 10 um. (C). Percentages of anaphase cells with lagging chromosomes (left graph) and of chromosomal bridges (right graph) in AUX- and AUX + cells (Total 34-104 anaphase cells per sample, n = 3 experiments). *p < 0.05. (paired t-test), NS; Non-Significant.

EWSR1 contributes to for Aurora B localization at inner centromere, and prevents its localization at kinetochore during early mitosis. (A). Representative images of Aurora B on pro/metaphase chromosome obtained from control (AUX-) cells that were released from thymidine/nocodazole for 30 min. The Aurora B (Green, top and bottom panel) and CENP-C (kinetochore marker) (Red, top and middle panel) were visualized by immunocytochemistry using anti-Aurora B and anti-CENP-C antibodies. Classification of the localization of Aurora B; Inner Centromere (IC), Kinetochore Proximal Centromere (KPC), Inner Centromere and Kinetochore Proximal Centromere (IC + KPC), and No Signal. Scale bar = 10 um. (B). Quantification of Aurora B localization at IC, KPC, IC + KPC, and No signal between AUX- and AUX + cells (total of 712–732 chromosomes per sample, n = 3 experiments). Values are mean with standard error of the mean (SEM). Two-tailed paired t-test; *p < 0.05, **p < 0.01, NS; Non-Significant.

EWSR1 partially colocalizes with CENP-C and Aurora B at the kinetochore proximal region in DLD-1 cells. (A) Merged images with CENP-C (Red), mNEON-EWSR1 (Green), and DNA stained using DAPI (blue). CENP-C (red) was visualized using an anti-CENP-C antibody, and mNEON-EWSR1 is shown in green (Left panel); CENP-C (Red) and mNEON-EWSR1 (Green), CENP-C (Red), mNEON-EWSR1 (Green), and DAPI (Right panel). (B) Single Z-section images with Aurora B (Red), mNEON-EWSR1 (Green), and DNA stained using DAPI (blue). Aurora B (red) visualized using anti- CENP-C antibody, and mNEON-EWSR1 (green) (Left panel); Aurora B (Red) and mNEON-EWSR1 (Green), Aurora B (Red), mNEON-EWSR1 (Green), and DAPI (Right panel). Scale bar 10 um.

The EWSR1 knockdown cells do not arrest at any mitotic stages. (A). The percentages of mitotic stage of AUX- and AUX + cells released for 0, 30, 60 and 90 min after the thymidine/nocodazole treatment (total of 83–173 cells per sample, n = 3 experiments). NS = non-significant. (B). Representative images of western blotting using anti-FLAG (top panel), anti-Cyclin B (middle panel) and anti-β actin (bottom panel) obtained from AUX- and AUX + cells released at 0, 30, 60, and 90 min after the thymidine/nocodazole treatment. *: Non-specific band. (C). Quantification of the levels of Cyclin B protein (normalized by β-actin) obtained from AUX- and AUX + cells (n = 3 experiments). Images of western blotting gel using anti-EWSR1 (top panel), anti-Cyclin B (middle panel), and anti-β-actin antibodies (bottom panel). Values are mean with S.D. using two-way ANOVA with Tukey’s multiple comparison test. NS: Non-Significant.

The EWSR1 knockdown promotes the induction of aneuploidy. (A). Schematic diagram for the mitotic synchronization of AID-EWSR1 knockdown (synchronized to mitosis using thymidine/nocodazole, and treated with AUX for 48 h concurrently) cells. The mitotic cells were harvested 30 min after released from the arrest. (B). Representative images of chromosomes visualized with anti-CENP-C (red), and anti-Topoisomerase II (green) obtained from AUX- and AUX + cells. Scale bar = 10 um. (C). The percentage of cells that displayed normal 46 numbers of chromosomes was decreased in AUX + cells compared to AUX-cells. 18-24 cells per sample were counted from n = 3 experiments (Total of n = 56 cells from AUX-, and of n = 62 cells from AUX+). Two-tailed paired t-test, *p < 0.05. (D). The percentage of of cells with each of the chromosome numbers in AUX− and AUX + cells n = 3 experiments.

The interaction between EWSR1 and Aurora B prevents the induction of aneuploidy. (A). The high incidence of aberrant chromosome numbers in AUX+/DOX-treated cells was rescued by the expression of EWSR1 overexpression (AUX+/DOX+) cells (28-59 cells per sample, n = 3 experiments). One-way ANOVA with Tukey multiple comparison test., **p < 0.01, *p < 0.05. NS = Non-significant. (B). The percentages of cells with each chromosome numbers in AUX-/DOX- (n = 122), AUX+/DOX- (n = 117), and AUX+/DOX+(n = 106) cells. (C). The high incidence of aberrant chromosome numbers in AUX+/DOX-cells was not rescued by the expression of EWSR1:R565A in AUX+/DOX + cells. (35-42 cells per sample, n = 6 experiments, One-way ANOVA with Tukey multiple comparison test. *p < 0.05, NS = non-significant. (D). The percentage of cells with each chromosome number in AUX-/DOX- (n = 221), AUX+/DOX- (n = 228), and AUX+/DOX+(n = 219) cells.

Schematic model of the study. The EWSR1 knockdown leads to the increased incidence of relocation of Aurora B from inner centromere to KPC (prometaphase and metaphase), of lagging chromosomes (anaphase), and of aneuploidy (after a single mitosis). Our data suggests that the EWSR1 prevents the induction of aneuploidy through interaction with Aurora B. Despite of the induction of chromosome mis-segregation in EWSR1 knockdown cells, the cells do not arrest at mitosis, and it is likely that the cells are overriding the error collection process.