FIG. 5.

Cyclin A-Mcm7 interaction is essential for S-phase entry. (a) Left panel: 293 cells were cotransfected with the vector expressing HA-tagged mouse cyclin A (HA-mCycA) and the indicated concentrations of siRNAs, and the cell lysates were subjected to anti-HA Western blot analysis. Right panel: NIH 3T3 cells stably expressing no exogenous cyclin (mock) or myc-tagged human wild-type (WT) or mutant (C1) cyclin A were transfected with the indicated amounts of si1 RNA, and the cell lysates were subjected to anti-cyclin A Western blot analysis. MTCycA, myc-tagged human cyclin A; endo CycA, endogenous mouse cyclin A. (b and c) The NIH 3T3 cell lines described for panel a were cotransfected with si1 RNA (0, 2, and 4 nM) and a GFP-expressing vector, in the absence (b) and presence (c) of the Mcm7-3 mutant-expressing vector. For panel c, pCS2HA was used for the control transfection without Mcm7-3. Twenty-eight hours after transfection, the cell cycle distributions of both GFP⁺ (transfected) and GFP⁻ (nontransfected) cells were analyzed by FACS. Values are ratios of the percentages of G₁-, S-, and G₂/M-phase cells in GFP⁺ cells relative to those for GFP⁻ cells. The significances of the differences between cyclin A-expressing cells and mock-expressing cells (b) and between Mcm7-3-transfected cells and mock-transfected cells (c) were analyzed by the Student t test (see Tables S3 and S4 in the supplemental material).