MYOCD Antibody - #DF2434

Fig. 4: SREBP1 directly promotes the senescence-like transformation of VSMC by activating lipogenesis. A Correlation between the expressions of SREBPs target genes and VSMC contractile genes (upper left panel), SREBPs target genes and aging genes (lower left panel), SREBP1 target genes and VSMC contractile genes (upper right panel), and SREBP1 target genes and aging genes (lower right panel) in the carotid artery from healthy and atherosclerosis patients (GSE100927). Correlation and P value (two-tailed) are based on Pearson’s correlation. B Heatmap depicting the average expression level of VSMC contractile genes, SREBP1 related genes, aging genes, and fibroblast genes of each VSMC or fibroblast subgroups in the carotid artery from atherosclerosis patients (single-cell transcriptomic, GSE253903). C Relative mRNA expression levels of genes involved in the maintain of VSMC contractile phenotype of MOVAS transfected with siCtrl, si-Srebf1#3, or si-Srebf2#3. Data are presented as mean ± SD. Statistical significance is calculated with a one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 6 biological replicates). D Representative images of cell contraction assay of MOVAS transfected with siCtrl, si-Srebf1#3, or si-Srebf2#3. E Schematic diagram of the agonistic target of LXR-623. F Immunoblot analyses of precursor-SREBP1/2 (p-SREBP1/2), and nucleic-SREBP1/2 (n-SREBP1/2) of MOVAS after DMSO or LXR-623 treatment. Band quantification is normalized to the first lane. G GSEA based transcription factors (TFs) enrichment analysis for the activated TFs of three RNA-seq datasets (CON vs. HU, CTRL vs. SM, and DMSO vs. LXR-623) is shown above. Details of the co-activated TFs (3 in total) are listed below. SREBP1 is highlighted. H GSEA plots showing normalized enrichment scores (NES) and normalized P-value (calculated with an empirical phenotype-based permutation test) for SREBP1 target genes from RNA-Seq (DMSO vs. LXR-623). I The key genes, regulatory processes (black arrows), and heatmap analysis (middle panel) involved in cholesterol synthesis (left panel) and fatty acid synthesis (right panel) pathways. The Log2-transformed fold change (FC) values are presented in parentheses. J Lipid level in MOVAS after DMSO or LXR-623 treatment by BODIPY staining. K Representative transmission electron microscopy images of MOVAS treated with DMSO or LXR-623. Lipid droplets are indicated by red arrows. Scale bar = 2 μm (left panel), 1 μm (zoom-in panel). L Representative images of the cell contraction assay of MOVAS treated with DMSO or LXR-623. Quantifications of cell contraction diameters are shown in the lower panel. Data are presented as mean ± SD. Statistical significance is calculated with a two-tailed unpaired t test (n = 5 biological replicates). M Immunoblot analyses of MYOCD, α-SMA, and SM22α of MOVAS treated with DMSO or LXR-623. Band quantification is normalized to the first lane. The non-specific bands are marked by “*”. N GSEA plots showing normalized enrichment scores (NES) and normalized P-value (calculated with an empirical phenotype-based permutation test) for VSMC contraction from RNA-Seq (DMSO vs. LXR-623). O Representative FACS images of MOVAS treated with DMSO or LXR-623 by SPiDER-βGal staining. P Representative images of β-Gal staining of MOVAS treated with H2O2 (positive control), DMSO or LXR-623. Scale bar = 200 μm.

FIGURE 3 MBNL1 increases Myocardin expression by binding to UGCU at the 3'-UTR of Myocardin mRNA. A, The 3'-UTR region of Myocardin contains the binding site of MBNL1. B, The binding of MBNL1 to Myocardin mRNA was validated using RIP. (n = 3, *, P < .05). C-F, The changes in Myocardin and MBNL1 in cardiomyocytes with overexpressed or silenced MBNL1 were measured using realtime PCR and Western blotting. (n = 3, *, P < .05, **, P < .01). G and H, Myocardin mRNA residues after 0, 20, 40 and 60 min in cardiomyocytes treated with Actinomycin D (ACD, 5 μg/mL) and with overexpressed or silenced MBNL1were measured using realtime PCR. I, The 3'-UTR region of Myocardin mRNA was transcribed in vitro and labelled with biotin. The combination of MBNL1 protein and 3'-UTR of Myocardin was detected using RNA pulldown assay. J, The RNA probes corresponding to nine prediction sites (probe # 10-18) and their corresponding antisense probes (probe # 1-9) were synthesized and biotin-labelled. The combination site of MBNL1 protein in the 3'-UTR of Myocardin was confirmed using RNA pull-down assay

Fig. 2. |Mhrt regulates myocardin expression through KLF4 in primary cardiomyocytes.(D, E and F) Following knock down of endogenous KLF4 in primary cardiomyocytes, the effect of Mhrt on myocardin mRNA and protein levels was examined (n = 3, *,P < .05, **, P < .01, #, P > .05).