Journal: Nature Communications

Article Title: The inner nuclear membrane protein LEMD3 organizes the 3D chromatin architecture to maintain vascular smooth muscle cell identity

doi: 10.1038/s41467-025-63876-3

Figure Lengend Snippet: a Schematic diagram showing the generation of an EGFP reporter-expressing MOVAS cell line. P2A, a self-cleaving peptide. b Representative flow cytometry and quantification of EGFP expression in the reporter MOVAS cell line in response to 48 h of TGF-β treatment (blue line), serum starvation (red line), or PDGF-BB treatment (green line). WT, wild-type; TGF-β, transforming growth factor-β; PDGF-BB, platelet-derived growth factor BB. n = 3 independent experiments. c Schematic diagram of the genome-scale CRISPR knockout screen. d Flow cytometry analysis of EGFP reporter knock-in MOVAS cells that were transduced with or without the pooled lentivirus encoding a genome-wide sgRNA library with two rounds of cell sorting. Wild-type (WT) MOVAS cells were utilized as a negative control for EGFP fluorescence. e Identification of the top candidate genes using the MAGeCK algorithm. Pink circles represent the top candidate genes that have been previously reported to regulate the VSMC phenotype. MAGeCK, Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout. RRA score, robust rank aggregation score. f RT‒qPCR analysis of Acta2 , Cnn1 , Tagln , and Myh11 gene expression in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 24 h. The data are presented as the relative fold changes to siRNA scramble (n = 5 independent experiments). Error bars indicate s.e.m. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test for panel b and two-sided unpaired Student’s t test for ( f ).

Article Snippet: The mouse aortic smooth muscle cell line MOVAS (CRL-2797), rat aortic smooth muscle cell line A7r5 (CRL-1444) and HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Flow Cytometry, Derivative Assay, CRISPR, Knock-Out, Knock-In, Transduction, Genome Wide, FACS, Negative Control, Fluorescence, Gene Expression, Transfection

Journal: Nature Communications

Article Title: The inner nuclear membrane protein LEMD3 organizes the 3D chromatin architecture to maintain vascular smooth muscle cell identity

doi: 10.1038/s41467-025-63876-3

Figure Lengend Snippet: a Representative images of immunocytochemical staining for LEMD3 in primary rat VSMCs. Scale bar = 100 µm. n = 3 independent experiments. b Representative images of immunohistochemical staining for LEMD3 in human internal mammary arteries. The region between the dashed lines corresponds to the medial area. Scale bar = 20 µm. n = 3 independent samples. c Representative images of immunofluorescence staining of rat VSMCs transfected with the pEGFP-N1-LEMD3 plasmid or control plasmid. The nuclei were stained with DAPI (blue). Scale bar = 5 µm. This experiment was repeated three times with similar results. d Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under serum starvation (48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to control (Ctrl) group (n = 6 independent experiments). e Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under rapamycin treatment (100 nM, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to vehicle group (n = 6 independent experiments). f Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under PDGF-BB treatment (10 ng/ml, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to vehicle group (n = 6 independent experiments). g Representative western blotting and quantification of LEMD3 in the lysates of sham-operated and wire-injured mouse carotid arteries at 3 days after surgery. GAPDH was used as an internal control. Data were presented as relative fold change to Sham (n = 4 independent mice). h Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, and TAGLN in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by serum starvation treatment (48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to siRNA scramble (n = 4 independent experiments). i Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, and TAGLN in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by rapamycin treatment (100 nM, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to siRNA scramble (n = 4 independent experiments). j Representative images of phalloidin staining and quantification of F-actin (red) in primary 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 = 25 µm. n = 3 independent experiments. k Collagen gel contraction assays using primary rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. n = 3 independent experiments. l EdU incorporation assays of primary rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. n = 6 independent experiments. Error bars indicate s.e.m. Statistical analysis was performed using two-sided unpaired Student’s t test for ( d-g ), two-way ANOVA followed by Bonferroni’s multiple comparisons test for ( h ) and ( i ), the χ 2 test for ( j ) and the Mann‒Whitney U test for ( k ) and ( l ).

Article Snippet: The mouse aortic smooth muscle cell line MOVAS (CRL-2797), rat aortic smooth muscle cell line A7r5 (CRL-1444) and HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC).

Techniques: Staining, Immunohistochemical staining, Immunofluorescence, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Nature Communications

Article Title: The inner nuclear membrane protein LEMD3 organizes the 3D chromatin architecture to maintain vascular smooth muscle cell identity

doi: 10.1038/s41467-025-63876-3

Figure Lengend Snippet: a Schematic diagram of the LEMD3 interactome. b Coimmunoprecipitation (Co-IP) assays of HEK293T cells cotransfected with LEMD3-FLAG and HA-CBX3 plasmids for 48 h. The lysates were immunoprecipitated with a control IgG antibody, anti-FLAG antibody, or anti-CBX3 antibody, followed by immunoblotting with the indicated antibodies. c Co-IP assays of endogenous LEMD3 and CBX3 in rat VSMCs. The lysates were immunoprecipitated with a control IgG antibody, anti-LEMD3 antibody, or anti-CBX3 antibody, followed by immunoblotting with the indicated antibodies. d Schematic illustration of LEMD3 domain depletion mutations used to evaluate the interaction with CBX3. The presence or absence of the deletion mutant binding to CBX3 was defined as + or −, respectively. e HEK293T cells were cotransfected with the CBX3 plasmid and the full-length LEMD3-FLAG, ΔC-terminal-FLAG, or ΔRRM-FLAG plasmid for 48 h. The cell lysates were immunoprecipitated with an anti-CBX3 antibody, and the precipitates were analyzed by immunoblotting with an anti-FLAG antibody. f Coimmunoprecipitation (Co-IP) assays of HEK293T cells cotransfected with RRM-FLAG and HA-CBX3 plasmids for 48 h. The lysates were immunoprecipitated with a control IgG antibody, or anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. g , h Representative western blotting and quantification of VSMC contractile marker expression in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by transfection with the pcDNA3.1, LEMD3-FLAG, or ΔRRM-FLAG plasmid for 48 h. GAPDH was used as an internal control. The data are presented as the relative fold changes compared with that of the pcDNA3.1+vehicle group (n = 5 independent experiments). i , j Representative western blotting and quantification of VSMC contractile marker expression in the A7r5 rat VSMC cell line transfected with the pcDNA3.1 or LEMD3-FLAG plasmid for 12 h, followed by transfection with the scrambled siRNA (20 nM) or siRNA targeting Cbx3 (20 nM) for 48 h. GAPDH was used as an internal control. The data are presented as the relative fold changes compared with that of the pcDNA3.1+siRNA scramble group (n = 3 independent experiments). Data shown in ( b, c, e and f ) are from one representative of three independent experiments with similar results. Error bars indicate s.e.m. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test for ( h ) and ( j ).

Article Snippet: The mouse aortic smooth muscle cell line MOVAS (CRL-2797), rat aortic smooth muscle cell line A7r5 (CRL-1444) and HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Mutagenesis, Binding Assay, Plasmid Preparation, Marker, Expressing, Transfection

Journal: Nature Communications

Article Title: The inner nuclear membrane protein LEMD3 organizes the 3D chromatin architecture to maintain vascular smooth muscle cell identity

doi: 10.1038/s41467-025-63876-3

Figure Lengend Snippet: 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 ).

Article Snippet: The mouse aortic smooth muscle cell line MOVAS (CRL-2797), rat aortic smooth muscle cell line A7r5 (CRL-1444) and HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Staining, Transfection, Fluorescence, Software, Western Blot, Control, ChIP-sequencing, Hi-C, Electron Microscopy

Journal: medRxiv

Article Title: Enhancer-targeting CRISPR screens at coronary artery disease loci suggest shared mechanisms of disease risk

doi: 10.1101/2025.08.28.25334684

Figure Lengend Snippet: A) Linkage disequilibrium (LD) for select 9p21.3 variants associated with CAD and calcification. LD for EUR alleles is shown for individual pairs of SNPs that have been identified as contributing to risk for CAD and vascular calcification, with YRI LD values indicated for highlighted SNPs shown in parentheses. B) AHR mediated transcriptional expression linked to rs1537372 with reporter gene studies in A7R5 rat SMC. C) Singe cell RNA sequencing data indicates an inhibitory role of Ahr toward expression of Cdkn2b and Cdkn2a in a mouse atherosclerosis disease model.

Article Snippet: Rat aortic smooth muscle cells (A7r5) and human embryonic kidney 293 cells (HEK293) were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Fisher Scientific, #MT10013CV) with 10% FBS at 37 °C and 5% CO2.

Techniques: Expressing, RNA Sequencing

Journal: medRxiv

Article Title: Enhancer-targeting CRISPR screens at coronary artery disease loci suggest shared mechanisms of disease risk

doi: 10.1101/2025.08.28.25334684

Figure Lengend Snippet: A) TF binding motifs and their relationship to variants at rs4977574. B) Transcriptional expression linked to rs4977574 with reporter gene studies in A7R5 rat SMC. DHT, dihydrotestosterone; PR, progesterone receptor; P4, progesterone; ERa, estrogen receptor alpha; E2, estradiol 1nM.

Article Snippet: Rat aortic smooth muscle cells (A7r5) and human embryonic kidney 293 cells (HEK293) were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Fisher Scientific, #MT10013CV) with 10% FBS at 37 °C and 5% CO2.

Techniques: Binding Assay, Expressing

Journal: medRxiv

Article Title: Enhancer-targeting CRISPR screens at coronary artery disease loci suggest shared mechanisms of disease risk

doi: 10.1101/2025.08.28.25334684

Figure Lengend Snippet: A) Heatmap shows AR binding regions across the human genome as determined with ChIP-seq in prostate carcinoma cells, colocalize with AHR binding sites in the same regions as identified with ChIP-seq of HCASMC (p <2.2e-16 by the Wilcoxon test). B) Violin plots of disruption scores show that variants in AR binding motifs are more likely to impair binding compared to other binding motifs of similar length and complexity such as shown for control TF HNF1A (p <2.2e-16) by the Wilcoxon rank sum test). C) Violin plots of disruption scores show that variants in AHR binding motifs are not more likely to impair binding compared to control GATA1 and ZNF354C TFs with similar length and complexity. D) AR and AHR cooperate to regulate transcription at AR binding sites, as shown for the rs4977574 AR binding site with luciferase reporter gene studies in A7R5 SMC. A luciferase reporter plasmid containing amplified DNA homozygous for the AR binding site at rs4977574 (A allele) was transfected into SMC with expression vectors encoding AHR, ARNT, AR, a combination of all TFs (A/A/A), AR with DHT stimulation (AR + DHT) or DHT stimulation alone with a control reporter construct.

Article Snippet: Rat aortic smooth muscle cells (A7r5) and human embryonic kidney 293 cells (HEK293) were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Fisher Scientific, #MT10013CV) with 10% FBS at 37 °C and 5% CO2.

Techniques: Binding Assay, ChIP-sequencing, Disruption, Control, Luciferase, Plasmid Preparation, Amplification, Transfection, Expressing, Construct