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. 2022 Feb 26;8:85. doi: 10.1038/s41420-022-00877-x

Fig. 4. DDK inhibition causes premature mitotic entry and mitotic catastrophe in Ewing sarcoma cells.

Fig. 4

A A673 and U2OS cells were treated with 0.1% DMSO, 1 µM XL413 or 300 nM TAK-931 for 24 h. Cells were then stained for phospho-histone H3 (Serine 10) (pHH3) and DNA (propidium iodide) and analyzed using FACS (representative dot plots of 3 biological replicates). B Quantification of panel A of proportion of pHH3-positive cells that had <4 N DNA content per total pHH3 cells (n = 3 biological replicates) 2-way ANOVA Dunnett’s multiple comparison test ****p < 0.0001. Bars represent mean and standard deviation. C Quantification of panel A of total pHH3 cells (n = 3 biological replicates) 2-way ANOVA Dunnett’s multiple comparison test *p = 0.0117. Bars represent mean and standard deviation. D A673 cells were treated with either 1 µM XL413 or 300 nM TAK-931 for 0, 24 or 48 h. Cells were then fixed and stained for DNA content (DAPI). Mitotic events were termed abnormal if they showed signs of anaphase bridge formation, lagging chromosome(s) during anaphase or anaphase/metaphase events with clear signs of more than 2 poles (n = 2 biological replicates) 2-way ANOVA **p < 0.01. Bars represent mean and standard deviation. Detailed quantitation and images can be found in Fig. S3E A673 cells were treated with 1 µM XL413 or 300 nM TAK-931 for 0, 24, 48 or 72 h. Cells were then fixed and stained for DNA content (DAPI). Micronuclei-containing cells were then quantified (n = 3 biological replicates) 2-way ANOVA ****p < 0.0001. Bars represent mean and standard deviation. F A673 cells were treated with 1 µM XL413 or 300 nM TAK-931 for 0, 24, 48 or 72 h. Cells were then fixed and stained for yH2AX, and total nuclear fluorescence intensity was calculated using FIJI image software. Representative quantification of 2 biological replicates 2way ANOVA, *p < 0.05, ****p < 0.0001. G Cells were treated with 1 µM XL413 (top row) or 300 nM TAK-931 (bottom row) for 0, 24, 48 or 72 h. Total DNA content was stained (PI) and % cells with <2 N DNA content (red) and >4 N DNA content (blue) were quantified using FACS (n = 3 biological replicates) Ordinary one-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001.