Figures & data
Figure 1. Effect of CycAΔ80 overexpression on cell cycle in human cells. (a) 10 µg each of myc-tagged cyclin A (mtCycA) WT or mtCycAΔ80 expression vector or empty vector (EV) was co-transfected with 1 µg of GFP expression vector in the indicated cell lines and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Arrows indicate the increase in G2/M populations. Cell cycle distributions of mtCycA WT- and mtCycAΔ80-transfected cells are shown in the bottom panel. **p < 0.01. (b) Extracts of the indicated cell lines were subjected to western analysis using antibodies against various CDK inhibitors and α-tubulin (internal control) (top). Protein amounts of p21, 27 and p107 relative to those of α-tubulin are shown (bottom). (c) Extracts from the indicated cells co-transfected with 10 µg of the mtCycA WT or Δ80 vector were immunoprecipitated with anti-myc tag, immunoblotted for mtCycA (top), and assayed for histone H1 kinase activity. H1 kinase activity (arbitrary units) relative to the amount of mtCycA proteins is shown in the bottom panel. (d) Indicated amounts of the expression vector for mtCycA WT, Δ80, or Δ80 R211A, which cannot bind to CDK, were co-transfected with 1 µg of the GFP vector in HEK293 cells and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top, left). Relative expression levels of exogenous CycA proteins are shown in the top right panel (maximum CycAΔ80 level = 1). Cell cycle distributions are shown in the bottom panel. *p < 0.05, **p < 0.01. (e) 2 µg of the mtCycAΔ80 vector was co-transfected with 1 µg of the GFP vector, and 4 µg of empty vector (0 µg CDK2) or indicated amounts of wild-type CDK2 (CDK2WT) or dominant-negative CDK2 mutant (CDK2dn) vector in HEK293 cells, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Cell cycle distributions are shown in the bottom panel. *p < 0.05.
Figure 1. Continued.
Figure 1. Continued.
Figure 2. G2/M-phase arrest is enhanced by co-expressing E1A. (a) Extracts from HEK293 cells were immunoprecipitated with control IgG and anti-E1A antibody, and immunoblotted for cyclin A (CycA), CDK1 and CDK2. (b) 10 µg of the mtCycAΔ80 vector was co-transfected with indicated concentrations of E1A-specific siRNA and 1 µg of the GFP vector in HEK293 cells, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Cell cycle distributions are shown in the bottom panel. **p < 0.01. (c) Extracts from HEK293 cells co-transfected with the mtCycAΔ80 vector and E1A-specific siRNA were immunoprecipitated with anti-CDK2, immunoblotted for E1A and mtCycAΔ80, and assayed for histone H1 kinase activity (left panel). Relative E1A protein amount (0 nM siRNA = 1) in the cell extracts (input) is shown in the top middle panel. H1 kinase activity relative to the amount of CDK2 proteins (0 nM E1A siRNA = 1) is shown in the top right panel. Relative amounts of CDK2-associated mtCycAΔ80 and E1A (0 nM E1A siRNA = 1) are shown in the middle and right bottom panels, respectively. *p < 0.05. (d) Indicated amounts of the mtCycAΔ80 vector was co-transfected with 5 µg of E1A expression vector (pE1A) and 1 µg of the GFP vector in MDAH041 cells, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Cell cycle distributions are shown in the bottom panel. *p < 0.05, **p < 0.01. (e) Extracts from MDAH041 cells co-transfected with 10 µg of the mtCycA WT or mtCycAΔ80 vector, with or without 5 µg of pE1A, were immunoprecipitated with anti-myc-tag and immunoblotted for CDK2 and E1A (left panel). Relative amounts of mtCycA-associated CDK2 (mtCycA WT only = 1) are shown in the right panel. **p < 0.01. (f) Extracts from MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 vector, with or without 5 µg of pE1A, were immunoprecipitated with anti-CDK2, immunoblotted for E1A, cyclin E, and mtCycAΔ80, and assayed for histone H1 kinase activity (left panel). H1 kinase activity relative to the amount of CDK2 proteins is shown in the middle top panel (E1A- = 1). Relative amounts of CDK2-associated mtCycAΔ80 and cyclin E (E1A- = 1) are shown in the bottom panels (middle and right, respectively).
Figure 2. Continued.
Figure 3. G2/M-phase arrest is induced by CycAΔ80 overexpression via ATR-Chk1 pathway. (a) MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 vector, 5 µg of pE1A and 1 µg of the GFP vector were treated with ATM/ATR, Chk1, and Chk2 inhibitors (final concentration: 4 mM caffeine, 1 μM MK8776, 50 nM UCN-01, 300 nM PV1019, respectively) for 24 h, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Cell cycle distributions are shown in the bottom panel. *p < 0.05, **p < 0.01. (b) MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 vector, 5 µg of pE1A and 1 µg of the GFP vector were treated for 24 h with 0.1 µM nocodazole added 16 h after transfection (①), then released from nocodazole for 8 h (②) or treated with 1 µM MK8776 for 8 h, before (③) or after (④) the release from nocodazole, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). As for the nocodazole-released condition (②), the cell cycle profile of the cells co-transfected with the mtCycA WT vector was also shown. Cell cycle distributions are shown in the bottom panel. **p < 0.01. (c) Extracts from MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 vector, with or without 5 µg of pE1A, were immunoprecipitated with anti-myc-tag and immunoblotted for the CDK1 and CDK1 phosphorylated at Y15 (pY15) (left). Relative amounts of mtCycA-associated CDK1-pY15 (E1A – = 1) are shown in the right panel. (d) Extracts from MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 and 5 µg of p E1A, treated for 24 h with 0.1 µM nocodazole 16 h after transfection and treated with or without 1 µM MK8776 for 8 h before harvest, were immunoprecipitated with anti-myc-tag and immunoblotted for the CDK1 phosphorylated at Y15 (pY15) and CDK1 (left). Relative amounts of mtCycA-associated CDK1-pY15 (-Chk1 inhibitor = 1) are shown in the right panel. *p < 0.05. (e) MDAH041 cells co-transfected with 10 µg of the mtCycAΔ80 vector, 5 µg of pE1A, 1 µg of the GFP vector and 3 µg each of empty vector (EV), wild-type CDK1 (CDK1WT), unphosphorylatable mutant CDK1 (CDK1AF) or cdc25A expression vector were treated for 24 h with 0.1 µM nocodazole after transfection, released for 8 h, and cell cycle profiles of the GFP-positive cells were analyzed by flow cytometry (top). Cell cycle distributions are shown in the bottom panel. *p < 0.05, **p < 0.01.
Figure 3. Continued.
Figure 3. Continued.
