Nucleic Acids Research, 2010, Vol. 38, No. 11 3541 Figure 5. Reduction of NRL in histone H1-null DT40 cells. Nuclei were isolated from DT40 cells and treated with MNase. DNA was then puriﬁed, separated on a 1% agarose gel, and stained with ethidium bromide. (A) Ethidium-stained agarose gel. Asterisks indicate the position of DNA fragments corresponding to penta-nucleosomes. (B) The size of DNA fragment corresponding to each number of nucleosomes was plotted and the NRL was calculated. Figure 6. Interphase nuclear volume is increased in H1-null cells. Three-dimensional images of DAPI-stained nuclei were collected using a confocal microscope, and nuclear volumes were measured and plotted. The mean values are indicated. The diﬀerence between the wild-type and H1 11/12 or H1 12/12 cells are signiﬁcant (P < 0.05, one-way ANOVA test). chromosomal aberrations in H1 12/12 #1 and #2 cells with that in wild-type, H1 11/12, H1 11/12+ﬂox and H1 12/ 12+ﬂox cells and categorized the chromosomal aberrations according to the International System for Human Cytogenetic Nomenclature (ISCN, 1985) (Table 1 and examples are shown in Supplementary Figure S2). As described previously (20), H1R-defective H1 11/12 cells exhibited a high chromosomal aberration rate [0.08 ± 0.014 aberrations per cell (apc) versus 0.02 ± 0.007 apc in wild-type cells] (20). The high chro- mosomal aberration rate was restored to the wild-type level (0.01 ± 0.004 apc) in H1 11/12+ﬂox cells that expressed additional H1R-eGFP. However, H1 12/ 12+ﬂox, which expresses only exogenous H1R-eGFP, exhibited higher genomic instability (0.05 ± 0.011 apc) than H1 11/12+ﬂox. Furthermore, both the histone H1-null mutant cells, H1 12/12 #1 and #2, exhibited the highest chromosomal aberration rates (0.15 ± 0.019 and 0.14 ± 0.025 apc, respectively), which were 7-fold higher than those of the wild type. Taken together, these results suggest that not only H1R, but also a certain amount of H1 (probably any H1 variant) is crucial for maintenance of chromosomal stability, because (i) histone H1 completely mutant cells showed the highest chromo- somal aberration rate and (ii) H1 12/12+ﬂox exhibited a higher chromosomal aberration rate than wild type,