Fig. 5: LEMD3 mediates heterochromatin perinuclear anchoring and maintains the global chromatin organization.

a Left, representative images of immunofluorescence staining for H3K9me3 (green) and Lamin B1 (red) in rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. DAPI staining (blue) indicates the nucleus. Scale bar = 10 µm. n = 3 independent experiments. Right, quantification of immunofluorescence staining intensities for H3K9me3. Upper, the intensity ratio of nuclear interior to periphery; Lower, total amount of nuclear intensities. Six cells were randomly selected in each experiment. n = 18 cells. b Fluorescence intensity along the white line in a single cell was measured using ImageJ software for the H3K9me3 (green) channel. Scale bar = 5 µm. This experiment was repeated three times with similar results. c Representative western blotting and quantification of H3K9me3 levels in rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. Histone H3 was used as an internal control. The data are presented as the relative fold changes to siRNA_scramble (n = 3 independent experiments). d ChIP-seq data (n = 2) showing the genomic occupancy of H3K9me3 in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. e, f Hi-C analysis (n = 2) of the A7r5 rat VSMC line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. e The 3D whole-nucleus maximum entropy model (line rendering) and plane model of the nucleus (bond rendering) of WT and Lemd3 KD VSMCs. Purple represents the A compartment, and green represents the B compartment. f Curve diagram showing the ratio of A/B compartments from the center to the periphery of the nuclei. Blue represents WT, and red represents Lemd3 KD. g Left, representative electron microscopy images of aortic smooth muscle cells from 12-week-old Lemd3^WT mice and Lemd3^SMKO mice. The yellow arrows mark the perinuclear heterochromatin. E elastic lamina. Scale bar = 2 μm. Right, Quantitative analysis of the width of perinuclear heterochromatin of aortic smooth muscle cells from 12-week-old Lemd3^WT mice and Lemd3^SMKO mice. Four vascular smooth muscle cells were randomly selected from each mouse aortic section. n = 6 mice per group. Error bars indicate s.e.m. Statistical analysis was performed using the Mann‒Whitney U test for (a) and (g), and two-sided unpaired Student’s t test for (c).