3540 Nucleic Acids Research, 2010, Vol. 38, No. 11 Figure 4. No signiﬁcant cell cycle defect on histone H1 mutants. (A) Mitotic chromosomes in H1 mutant cells. The wild-type and H1 mutant DT40 cells were stained with DAPI (cyan) and anti-a-tubulin followed by Cy3-conjugated anti-mouse IgG (red). Little diﬀerence was seen in metaphase chromosomes among the wild-type, H1 11/12+ﬂox (expressing 02H1 and H1R-eGFP) and H1 12/12 (H1-null) cells. (B) Interphase nuclei. Little diﬀerence was seen in global structure of interphase nuclei among the wild-type, H1 11/12 (expressing only 02H1) and H1 12/12 (H1-null); however, the nuclear volume was altered (Figure 6). (C) Cell-cycle analysis of the wild-type and H1 12/12 (H1-null) cells by ﬂow cytometry. Cells were pulse-labeled with BrdU for 10 min, ﬁxed, and stained with FITC-anti-BrdU to monitor DNA synthesis (vertical axis; log scale) and propidium iodide (PI) to detect DNA content (horizontal axis; linear scale). The cell cycle distributions were similar between two cell types. R2:G1 phase, R3:S phase, R4:G2-M phase, R5: sub-G1 phase. (D) Mitotic index of the wild-type and H1-null cells with or without DNA damage. H3S10ph-positive cell fractions in the absence (left) or presence (right) of 0.1 mg/ml colcemid (0–4 h) were monitored by ﬂow cytometry. G2/M checkpoint activation was examined by ionizing radiation (2.3 Gy).