Figure 6.
Figure 6. SET depletion delays the occurrence of cohesion fatigue and reduces Sgo1 relocation from inner centromeres to kinetochores in MG132-arrested cells. (A) Lysates of HeLa Tet-On cells with mock treatment or transfected with distinct SET siRNAs (2 and 4) were resolved with SDS-PAGE and blotted with the indicated antibodies. (B) HeLa Tet-On cells with mock or siSET treatment were arrested with MG132 for the indicated time. Three types of chromosome morphology were observed: I, chromosomes with two sister centromeres cohesed; II, chromosomes with two sister centromeres separated but two sister chromatids still paired; and III, chromosomes with sister chromatids scattered. Category I was defined as unseparated chromosomes, and categories II and III were defined as separated chromosomes. Quantification of mitotic cells with unseparated and separated chromosomes is shown. The average and standard deviation from three independent experiments are shown. At least 20 mitotic cells were examined for each condition. *, P < 0.05; ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (C) HeLa Tet-On cells transiently transfected with vectors or plasmids containing GFP-SET WT or 3K were further treated with SET siRNAs. Cells were treated with MG132 for 2 h before being subjected to chromosome spread. Quantification of unseparated (U) and separated (S) sister chromatids, described in B, is shown here. At least 35 mitotic cells were examined for each condition. The average and standard deviation from two independent experiments are shown here. **, P < 0.01. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (D) Representative images of MG132 (2 h)–arrested HeLa Tet-On cells with mock treatment or transfected with SET siRNAs. The outlined regions are amplified and shown in the right panel. The plotted curves define the relative localization of ACA (red) and Sgo1 (green) signals. Two types of localization patterns were identified and described in the main text. I, kinetochore localization; II, inner-centromeric localization. (E) Quantification of chromosomes with distinct Sgo1 localization patterns (I and II) in B. Only cells with intact centromeric cohesion were counted. The average and standard deviation from three independent experiments are shown. 50–80 kinetochores (five per cell) were examined for each condition. ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (F) Quantification of chromosomes with distinct Sgo1 localization patterns (I and II) in MG132 (1.5 h)–arrested HeLa Tet-On cells transfected with vectors (V) or plasmids containing GFP-SET WT or 3K. Only cells with intact centromeric cohesion were counted. The average and standard deviation from three independent experiments are shown. At least 45 kinetochores (five per cell) were examined for each condition. ***, P < 0.001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. n.s., not significant.

SET depletion delays the occurrence of cohesion fatigue and reduces Sgo1 relocation from inner centromeres to kinetochores in MG132-arrested cells. (A) Lysates of HeLa Tet-On cells with mock treatment or transfected with distinct SET siRNAs (2 and 4) were resolved with SDS-PAGE and blotted with the indicated antibodies. (B) HeLa Tet-On cells with mock or siSET treatment were arrested with MG132 for the indicated time. Three types of chromosome morphology were observed: I, chromosomes with two sister centromeres cohesed; II, chromosomes with two sister centromeres separated but two sister chromatids still paired; and III, chromosomes with sister chromatids scattered. Category I was defined as unseparated chromosomes, and categories II and III were defined as separated chromosomes. Quantification of mitotic cells with unseparated and separated chromosomes is shown. The average and standard deviation from three independent experiments are shown. At least 20 mitotic cells were examined for each condition. *, P < 0.05; ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (C) HeLa Tet-On cells transiently transfected with vectors or plasmids containing GFP-SET WT or 3K were further treated with SET siRNAs. Cells were treated with MG132 for 2 h before being subjected to chromosome spread. Quantification of unseparated (U) and separated (S) sister chromatids, described in B, is shown here. At least 35 mitotic cells were examined for each condition. The average and standard deviation from two independent experiments are shown here. **, P < 0.01. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (D) Representative images of MG132 (2 h)–arrested HeLa Tet-On cells with mock treatment or transfected with SET siRNAs. The outlined regions are amplified and shown in the right panel. The plotted curves define the relative localization of ACA (red) and Sgo1 (green) signals. Two types of localization patterns were identified and described in the main text. I, kinetochore localization; II, inner-centromeric localization. (E) Quantification of chromosomes with distinct Sgo1 localization patterns (I and II) in B. Only cells with intact centromeric cohesion were counted. The average and standard deviation from three independent experiments are shown. 50–80 kinetochores (five per cell) were examined for each condition. ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (F) Quantification of chromosomes with distinct Sgo1 localization patterns (I and II) in MG132 (1.5 h)–arrested HeLa Tet-On cells transfected with vectors (V) or plasmids containing GFP-SET WT or 3K. Only cells with intact centromeric cohesion were counted. The average and standard deviation from three independent experiments are shown. At least 45 kinetochores (five per cell) were examined for each condition. ***, P < 0.001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. n.s., not significant.

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