Journal: Cell reports
Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms
doi: 10.1016/j.celrep.2024.114527
Figure Lengend Snippet: (A–D) To assess the binding of LRP1-ICD (A), and recruitment of epigenetic repressors DNMT1 (B), EZH2 (C), or MBD2 (D), to the promoter regions of DKK1 , SPARC , and TIMP2 in EME6/7t EECs treated with ASC-CM, ChIP-qPCR was used at the indicated time points (0, 6, 24, 48, 96, and 168 h). n = 3 technical replicates. Data are represented by mean ± SD. p values were determined using ANOVA test. (E) Bisulfite pyrosequencing analysis targeting the promoter regions of DKK1 , SPARC , and TIMP2 to assess CpG methylation levels in EME6/7t EECs exposed to ASC-CM (experimental duplicates: CM1 and CM2) or control (experimental duplicates: C01 and C02) for 21 days. The green box indicates CpG island, and the short, thick black line over the gene body represents the scale of 200 bp. The thicker, short blue box represents the probed sites. (F) Model for the underlying repressive actions by ASC-CM, which leads to the sequential recruitment of epigenetic repressors and subsequently marks the promoters of DKK1 , SPARC , and TIMP2 for methylation. LRP1-ICD initiates transcriptional repression of these genes. EZH2 and DNMT1 binding then enforces DNA methylation and establishes epigenetic silencing. See also – , and .
Article Snippet: Sonicated DNA fragments (~300–500 bp) were incubated with rabbit monoclonal anti-LRP1-ICD antibody (Abcam, Cat# ab92544), mouse monoclonal anti-YAP1 antibody (Novus Biologicals, Cat# NBP2–22117), rabbit monoclonal anti-Pan-TEAD antibody (Cell Signaling Technology, Cat# 13295S), mouse monoclonal anti-EZH2 antibody (Active Motif, Cat# 39076), goat polyclonal anti-MBD2 antibody (Novus Biologicals, Cat# NB100–55415), mouse monoclonal anti-DNMT1 antibody (Novus Biologicals, Cat# NB100–56519), normal goat IgG antibody (Abcam, Cat# ab37373), normal mouse IgG antibody (Millipore, Cat# NI03–100UG) or normal rabbit IgG antibody (Pierce Magnetic ChIP Kit, ThermoFisher Scientific, Cat# 26157) overnight at 4°C.
Techniques: Binding Assay, ChIP-qPCR, CpG Methylation Assay, Control, Methylation, DNA Methylation Assay