Figure 5.
Figure 5. SET overexpression accelerates the occurrence of cohesin fatigue in MG132-arrested cells. (A) Lysates of RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K were resolved with SDS-PAGE and blotted with the indicated antibodies. (B) SET overexpression barely affects centromeric cohesion in nocodazole-arrested cells. Nocodazole-arrested RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K were subjected to chromosome spread. Quantification of unseparated (blue bars) and separated (red bars) sister centromeres is shown here. The average from two independent experiments is shown here. (C) Overexpression of SET WT, not 3K, induces significant centromeric cohesion defects in MG132-arrested RPE-1 cells. Shown here are representative images of chromosome spread from MG132 (3 h)–arrested RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K. The outlined regions showing unseparated (V and 3K) and separated (WT) sister centromeres are amplified and shown in the bottom panel. (D) Quantification of mitotic cells in C with separated centromeres. The average and standard deviation from three independent experiments are shown here. At least 30 cells were examined for each condition. **, P < 0.01. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. n.s., not significant. (E) Overexpression of SET WT induces cohesion fatigue in MG132-arrested HeLa Tet-On cells. MG132 (1.5 h)–treated HeLa Tet-On cells transfected with vectors or plasmids containing GFP-SET WT, 3K, ΔC, or 3K-ΔC were subjected to chromosome spread. Quantification of mitotic cells with separated and unseparated sister chromatids is shown here. The relative intensity was derived from the intensity of Sgo1 signals normalized to one of the ACA signals. At least 60 kinetochores (five per cell) were examined for each condition. The average and standard deviation from at least three independent experiments are shown here. ***, P < 0.001; ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (F) Cell lysates in E were resolved with SDS-PAGE and blotted with the indicated antibodies.

SET overexpression accelerates the occurrence of cohesin fatigue in MG132-arrested cells. (A) Lysates of RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K were resolved with SDS-PAGE and blotted with the indicated antibodies. (B) SET overexpression barely affects centromeric cohesion in nocodazole-arrested cells. Nocodazole-arrested RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K were subjected to chromosome spread. Quantification of unseparated (blue bars) and separated (red bars) sister centromeres is shown here. The average from two independent experiments is shown here. (C) Overexpression of SET WT, not 3K, induces significant centromeric cohesion defects in MG132-arrested RPE-1 cells. Shown here are representative images of chromosome spread from MG132 (3 h)–arrested RPE-1 cells transfected with vector control (V) or plasmids containing GFP-SET WT or 3K. The outlined regions showing unseparated (V and 3K) and separated (WT) sister centromeres are amplified and shown in the bottom panel. (D) Quantification of mitotic cells in C with separated centromeres. The average and standard deviation from three independent experiments are shown here. At least 30 cells were examined for each condition. **, P < 0.01. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. n.s., not significant. (E) Overexpression of SET WT induces cohesion fatigue in MG132-arrested HeLa Tet-On cells. MG132 (1.5 h)–treated HeLa Tet-On cells transfected with vectors or plasmids containing GFP-SET WT, 3K, ΔC, or 3K-ΔC were subjected to chromosome spread. Quantification of mitotic cells with separated and unseparated sister chromatids is shown here. The relative intensity was derived from the intensity of Sgo1 signals normalized to one of the ACA signals. At least 60 kinetochores (five per cell) were examined for each condition. The average and standard deviation from at least three independent experiments are shown here. ***, P < 0.001; ****, P < 0.0001. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. (F) Cell lysates in E were resolved with SDS-PAGE and blotted with the indicated antibodies.

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