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.