Quantitative Mcm Perturbation in Mice Figure 7. MCM3 regulates nuclear and chromatin-bound MCM levels. (A) Predicted nuclear export sequences (NES) in mouse MCM3 (see text). (B) Western blot analysis of MCM2/3/4 in the indicated genotypes of MEFs. Three different protein fractions were examined, with the indicated (arrow) loading controls at the bottom. (C) qRT-PCR analysis of Mcm2-7 mRNAs in MEFs of the indicated genotypes. (D) Nuclear MCM2 dynamics during the cell cycle. The flow plot is of isolated nuclei stained for DNA content (PI = propidium iodide) on the X-axis, and MCM2 on the Y-axis. NIH3T3 cells show dramatically the decrease in nuclear MCM2 through S phase. Flow cytometric data from the 4 MEF genotypes indicated in the right panel were used to calculate two values, DG1 and DS. The regions for the calculation of these values are indicated, and the values plotted in the right panel. The G1 (1N DNA content) phase nuclei were divided into two equal groups based on MCM2 signal intensity (Y-axis): the lower half, considered to be early-G1, and the upper half, considered to be late-G1. The DG1 value was calculated as the difference between the early and late MCM2 signal intensity averages. The DS value was calculated as : (average MCM2 intensity in the S population) – (early G1 average intensity). C3 = Mcm4Chaos3; M = Mcm. C3 is set at 100%. The asterisk indicates significance by Student’s t-test (P,0.05). doi:10.1371/journal.pgen.1001110.g007 PLoS Genetics | www.plosgenetics.org 11 September 2010 | Volume 6 | Issue 9 | e1001110