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. 2019 Nov 25;19(1):84–96. doi: 10.1080/15384101.2019.1693119

Figure 3.

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.