Fig 1.
Generation of recombinant CP-Geminin protein
(A) Structure of CP-Geminin. Amino acid numbers indicate location of the representative domains. The coiled-coil domain is required for the dimerization or multimerization and also for the interaction with Cdt1. MTM: membrane translocating motif from FGF4. (B) Production and affinity-purification of recombinant CP-Geminin. Proteins were separated on SDS-PAGE and subjected to CBB staining. MW: molecular weight.
Fig 2.
Geminin transduction by means of CP-Geminin
(A) CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin and Geminin were added into the medium. One and 12h after the addition, cells were observed under a confocal microscope. The nucleus was stained with Hoechst33342. DIC: differential interference contrast (B) FITC-conjugated CP-Geminin incorporation into leukemic cell lines 24 h after the addition. (C) Dose dependency of CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin was added to the medium at the indicated concentration, and 12h after the addition, the cells were examined under a confocal microscope. (D) Withdrawal study of CP-Geminin. FITC-conjugated CP-Geminin was added to the medium and the incorporation was confirmed 12h after the addition. CP-Geminin was then removed by replacing the medium with CP-Geminin with one without CP-Geminin. One and 4h after the removal, the cells were examined under a confocal microscope.
Fig 3.
Molecular interaction of incorporated CP-Geminin with the target substrates.
Expression plasmid vectors for Myc-Cdt1, HA-Brahma-domainII and HA-Brg1-domainII were transfected into NIH 3T3 cells, after which CP-Geminin was added. The cells were then subjected to immunoprecipitation analysis. 1. Control, 2. Flag-Geminin, 3. CP-Geminin including a Flag tag.
Fig 4.
Effect of CP-Geminin on chromatin configuration and transcription.
(A) Effect of CP-Geminin on nuclease accessibility of the E2F-R chromatin locus of the Geminin gene. Twenty-four h after transduction of CP-Geminin (1,000 nM), cells were subjected to nuclease accessibility analysis. BSA, control (B) Effect on transcription of the Geminin gene promoter. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 μg) and the luciferase reporter plasmids (1.5 μg) were co-transfected, and transcription activity of the Geminin gene promoter was examined with the luciferase reporter assay. (C) Effect of CP-Geminin on mRNA expression of the endogenous genes, Geminin, Mcm7, CcnA2 and Actb2. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 μg) was transfected, and cells were subjected to TaqMan real-time PCR analysis an additional 24h after the transfection. *: P< 0.01.
Fig 5.
Biological function of CP-Geminin.
(A) CP-Geminin was transduced into synchronized NIH 3T3 cells by means of serum depletion, and its effect on the cell cycle was observed until 72 h after serum induction. (B) Cell cycle status 16h after serum induction. S-phase progression was markedly suppressed by CP-Geminin. *: P< 0.01.