Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Weighted gene coexpression network analysis of the GSE153434 dataset and Venn diagram to obtain two crucial genes. (A) A soft threshold power value of 8 was used, which is the optimum option. (B) Demonstration of scale‐free network validation with a soft threshold of 8. (C) By clustering genes with strong correlations into the same module, different modules were generated. Different modules are displayed in different colors. (D) Network heatmap showing branching of overall genes associated with modules in a hierarchical clustering dendrogram. (E) Analysis of the correlation between each module and TAAD. (F) The black module was significantly positively correlated with TAAD (correlation coefficient = 0.94, p < 0.001). (G) Venn diagram showing the intersection of DEGs obtained after RRA analysis, the WGCNA hub genes, and the TAAD‐related genes in GeneCards, finally yielding TIMP1 and LDB3 as the crucial genes.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Single‐cell sequencing analysis in the GSE213740 dataset. (A) Cell sample quality was assured by the analysis of three metrics: RNA count, gene count, and mitochondrial gene percentage. (B) The 2000 most highly variable genes are shown in red, with the top 10 highly variable genes marked. (C) Dimensionality reduction and clustering analysis were performed on the cells in the dataset, and the clustering of the TAAD and control groups is displayed in the ‘tSNE’ diagram. (D) Annotation of the clustered cells, including T cells, smooth muscle cells, fibroblasts, macrophages, mesenchymal cells, endothelial cells, monocytes, plasmocytes, giant cells, and B cells. (E) Expression of TIMP1 and LDB3 in cell clusters.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Sequencing, Control, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Pseudochronological analysis and pathway enrichment analysis of smooth muscle cells in the GSE213740 dataset. (A) Smooth muscle cells in the dataset were extracted and reclustered into nine clusters. (B) Expression analysis of LDB3 in smooth muscle cells from the GSE213740 dataset. (C) Pseudotime analysis of smooth muscle cells. The three images in the illustration show the three different differentiation states based on the aggregation of nine cell clusters and the differences in the time series of cell differentiation. (D) GO enrichment analysis of significant DEGs between cell clusters 1 and 5 (with considerable LDB3 expression) and the remaining seven cell clusters (with insignificant LDB3 expression). (E) KEGG enrichment analysis based on significantly differentially expressed genes between cell clusters 1 and 5 and the remaining seven cell clusters.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Expressing, Cell Differentiation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: LDB3 SNPs analysis in the UK Biobank. Univariate (19 SNPs) and multivariate (rs34346901 and rs117443987) associations between SNPs and aortic aneurysm and dissection. Six SNPs were significantly related, and the relative ratios of each genotype for aortic aneurysm and dissection are shown in the pie charts.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Dissection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Validation of LDB3 in human samples and upregulation of LDB3 by Ang II stimulation in HA‐VSMC. (A) Histology staining of TAAD and normal tissue. HE, Masson's trichrome staining, and EVG staining were used to reveal the differences in cellularity, collagen fibers, and elastic fibers in ascending aorta sections of TAAD and normal tissues. (B) Tissue collagen and elastin quantification ( n = 10). (C) Immunofluorescence staining showed that the expression of LDB3 was significantly decreased in TAAD vascular smooth muscle cells. (D, E) Long and short isoforms of LDB3 were expressed in TAAD aortic wall samples, and Western blot verification revealed that both long and short LDB3 expressions were significantly downregulated in TAAD compared to normal samples ( n = 10). (F, G) Ang II induced HA‐VSMC significant bands of LDB3 and quantification analysis. (H, I) Immunofluorescence staining of LDB3 and phalloidin in HA‐VSMC after Ang II treatment and quantification analysis ( n = 4). * p < 0.05, ** p < 0.01, **** p < 0.0001 in comparisons of two groups as indicated or compared with the corresponding control.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Staining, Immunofluorescence, Expressing, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Validation of LDB3 expression in Ang II‐induced aortic dissection aneurysm models. (A) Representative photographs showing macroscopic features of dissection aneurysms induced by Ang II. (B, C) Representative ultrasound images of aortic dimension in two groups and quantification analysis ( n = 5). (D, E) HE, Masson's trichrome staining, and EVG staining and elastin degradation score analysis ( n = 5). (F, G) Immunofluorescence staining of LDB3 in Ang II‐induced aortic dissection aneurysm and its quantification analysis ( n = 5). * p < 0.05, ** p < 0.01, **** p < 0.0001 in comparisons of two groups as indicated or compared to the corresponding control.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Expressing, Dissection, Staining, Immunofluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy

doi: 10.3390/ijms25126547

Figure Lengend Snippet: Family pedigree with black symbols indicating the affected members. The arrow indicates the proband ( A ). Electropherograms in the proband, his sister, and their father ( B ). Representative Western blot of controls and patient samples. Lane 1–4 staining with LDB3 (78 KDa and 32 KDa) and desmin. Lane 5–8 staining with LDB3 and myotilin. α-actinin-1 was used as loading control ( C ). Schematic representation of LDB3 protein structure with the indication of the already reported variants. The arrow shows the localization of our patient’s mutation in the PDZ domain ( D ).

Article Snippet: Membranes were probed with antibodies to LDB3 (1:15,000, goat polyclonal; ab110003 Abcam, Cambridge, UK), to myotilin (1:250, mouse monoclonal; Novocastra, Newcastle upon Tyne, UK), to desmin (1:200, mouse monoclonal; Novocastra, Newcastle upon Tyne, UK), and α-actinin-1 (1:5000; Sigma-Aldrich, Burlington, MA, USA). α-actinin-1 was used as an indicator of protein loading.

Techniques: Western Blot, Staining, Mutagenesis

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: (A) Overall scheme of disease-specific phosphorylated site selection. Only 47 phosphoproteins among 134 upregulated phosphoproteins were identified as phosphoproteins related to heart and muscle disease in the human disease database. Twenty-nine phosphoproteins were mapped in the heart tissue-specific expression database and literature. The changes at each phosphorylation site were compared with its protein abundance to identify phosphorylation-specific level changes. Finally, 14 target phosphoproteins and phosphorylation competition peptides for 18 phosphorylation target sites were selected for cell-based screening. (B) Cultured neonatal rat cardiomyocytes were treated with 100 nM competition peptides to inhibit site-specific phosphorylation and exposed to 100 µM PE for 24 h. The cell surface area was then determined by immunofluorescence staining of α-actinin, and bar graphs representing surface area were drawn. Statistical significance was assessed by two-tailed Student’s t -tests for each comparison group with three biological replicates. (C) Immunofluorescence images of sarcomeric α-actinin (a, untreated; b, PE; c, TAT; d, TAT + PE; e, Ldb3-S98 + PE; f, Ldb3-S121, 123 + PE; g, Ldb3-S170, 171 + PE; h, Ldb3-S179 + PE; i, Palld-S901 + PE; j, Pallad-S1146 + PE). PE, phenylephrine; TAT, transactivator of transcription. (D) Bar graphs showing changes in cell surface area following treatment with different concentrations of competition peptides targeting Ser-98 and Ser-179 of Ldb3. *** P < 0.001 vs untreated; ## P < 0.01, ### P < 0.001 vs TAT + PE; n.s., not significant; n = 3 per group.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Selection, Expressing, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Heart lysates were immunoblotted with non-phospho-antibody, Ser-98 site-specific phospho-antibody, and Ser-179 site-specific phospho-antibody in (A) 8-week-old and (B) 12-week-old JTT-1 TG mice. Bar charts show the relative intensity of site-specific phosphorylation compared with wild-type expression. P values determined using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Expressing, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Changes in site-specific phosphorylation of Ser-98 and Ser-179 of Ldb3 were investigated in (A) physiological cardiac hypertrophy (swimming training for 2 or 4 weeks), and (B) pathological cardiac hypertrophy (TAC and sham surgery for 2 weeks). Lysates were probed with non-phospho- and phospho-antibodies (recognizing Ser-98 or Ser-179 of Ldb3) to detect changes in endogenous site-specific phosphorylation. * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. con, control; sw, swimming.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: