Figure S3.
Rescue of cell cycle defects caused by cyclin B deficiency with cyclin B1 and cyclin A. (A) Expression of cyclin B1-YFP in cyclin B-deficient cells. mAIDB1KOB1B2 cells were transfected with a control plasmid or plasmids expressing YFP or cyclin B1-YFP. At 16 h after transfection, the cells were treated with DI and harvested at different time points. Lysates were prepared and analyzed with immunoblotting. (B) Ectopic expression of cyclin B1 rescues cell cycle defects induced by cyclin B deficiency. mAIDB1KOB1B2 cells transfected with plasmids expressing YFP or cyclin B1-YFP were treated with DI and harvested at the indicated time points for flow cytometry analysis. The DNA profiles of transfected YFP-positive cells are shown. (C) Overexpression of cyclin A in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing either mRFP or cyclin A-mRFP were treated with DI and harvested at specific time points. Lysates were prepared and analyzed with immunoblotting. (D) Overexpression of CDK1 and CDK2 in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing mRFP, CDK1-mRFP, or CDK2-mRFP were treated with DI and harvested at specific time points for immunoblotting analysis. (E) Ectopic expression of cyclin A promotes DNA re-replication in cyclin B-deficient cells. mAIDB1KOB1B2 cells were transfected with plasmids expressing mRFP or mRFP-tagged CDK1, CDK2, or cyclin A, followed by treatment with buffer or DI for 48 h. The cells were harvested and analyzed with flow cytometry. The DNA profiles of transfected mRFP-positive cells are shown. The asterisk indicates the population containing >4N DNA content. (F) Doubling cyclin A expression is insufficient to restore normal mitosis in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing “low” level of cyclin A and “high” level of cyclin A (see Fig. 6 A) were treated and imaged as described in Fig. 6 D. Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. (G) Localization of cyclin A-mRFP to both nucleus and cytoplasm during interphase. mAIDB1KOB1B2 cells expressing “high” level of cyclin A were treated and imaged as described in Fig. 6 D. Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. Source data are available for this figure: SourceData FS3.

Rescue of cell cycle defects caused by cyclin B deficiency with cyclin B1 and cyclin A. (A) Expression of cyclin B1-YFP in cyclin B-deficient cells. mAIDB1KOB1B2 cells were transfected with a control plasmid or plasmids expressing YFP or cyclin B1-YFP. At 16 h after transfection, the cells were treated with DI and harvested at different time points. Lysates were prepared and analyzed with immunoblotting. (B) Ectopic expression of cyclin B1 rescues cell cycle defects induced by cyclin B deficiency. mAIDB1KOB1B2 cells transfected with plasmids expressing YFP or cyclin B1-YFP were treated with DI and harvested at the indicated time points for flow cytometry analysis. The DNA profiles of transfected YFP-positive cells are shown. (C) Overexpression of cyclin A in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing either mRFP or cyclin A-mRFP were treated with DI and harvested at specific time points. Lysates were prepared and analyzed with immunoblotting. (D) Overexpression of CDK1 and CDK2 in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing mRFP, CDK1-mRFP, or CDK2-mRFP were treated with DI and harvested at specific time points for immunoblotting analysis. (E) Ectopic expression of cyclin A promotes DNA re-replication in cyclin B-deficient cells. mAIDB1KOB1B2 cells were transfected with plasmids expressing mRFP or mRFP-tagged CDK1, CDK2, or cyclin A, followed by treatment with buffer or DI for 48 h. The cells were harvested and analyzed with flow cytometry. The DNA profiles of transfected mRFP-positive cells are shown. The asterisk indicates the population containing >4N DNA content. (F) Doubling cyclin A expression is insufficient to restore normal mitosis in cyclin B-deficient cells. mAIDB1KOB1B2 cells expressing “low” level of cyclin A and “high” level of cyclin A (see Fig. 6 A) were treated and imaged as described in Fig. 6 D. Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. (G) Localization of cyclin A-mRFP to both nucleus and cytoplasm during interphase. mAIDB1KOB1B2 cells expressing “high” level of cyclin A were treated and imaged as described in Fig. 6 D. Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. Source data are available for this figure: SourceData FS3.

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