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. 2020 Nov 5;13(4):258–280. doi: 10.1007/s13238-020-00794-8

Figure 1.

Figure 1

Lamin B1 regulates chromatin subnuclear localization and global compaction. (A) Immunostaining of H3K27me3. Red: H3K27me3. Green: lamin B1. Blue: DAPI staining. The 2D sections of nuclei are displayed. Scale bars, 5 µm. Fluorescence intensity along the white line was measured using ImageJ software for H3K27me3 and DAPI channels. (B) Normalized fluorescence intensity of H3K27me3 in each shell in WT (n = 35) and lamin B1-KO (n = 39) cells. 2 independent experiments. Mean + standard deviation (SD). P < 0.05, paired t test. (C) Immunostaining of H3K4me2, H3K4me3 and H3K27ac. Red: histone modifications. Green: lamin B1. Blue: DAPI staining. The maximum intensity projections of nuclear Z stacks are displayed. Scale bars, 5 µm. (D) Normalized total fluorescence intensity of immunostaining signals of H3K4me2, H3K4me3 and H3K27ac. Statistical analysis shows increased level of three histone modifications in lamin B1-KO cells. ***P < 0.001, paired t test. 2 independent experiments. (E) Representative 3D-projection and 3D-reconstruction chromosome painting images of chromosome 2 and 18. Green: FISH signals of chromosome 2. Red: FISH signals of chromosome 18. Blue: DAPI staining. For 3D-projection images, the maximum intensity projections of nuclear Z stacks are displayed, Scale bars, 5 µm. 3D-reconstruction images are processed in Imaris software, Scale bars, 2 µm. (F) Quantification of the nuclear localization of chromosomes based on their relative distances from the chromosome mass center to the nuclear mass center. This distance is normalized by the cubic root of the nuclear volume. Mean ± SD. ***P < 0.001, Mann-Whitney test. 3 independent experiments. (G) Quantification of the volumes occupied by chromosome 2 and 18 relative to the nuclear volume. Chromosomes in lamin B1-KO cells show significantly larger relative volumes. Mean ± SD. ***P < 0.001, Mann-Whitney test. 3 independent experiments. (H) Quantification of the overlap frequency between chromosome 2 and chromosome 18 territories. The ratio of cells presenting territory interaction between chromosome 2 and chromosome 18 in WT cells is significantly smaller than that in lamin B1-KO cells. ***P < 0.001, Fisher’s exact test. 3 independent experiments