Journal: Cells

Article Title: Integrated Analysis of DNA Methylome and Transcriptome Reveals Regulatory Mechanism in the Longissimus Dorsi of Duroc Pigs

doi: 10.3390/cells14110786

Figure Lengend Snippet: DNA methylation regulates gene expression by regulating the accessibility of TF binding: ( A ) Relative dual luciferase report for adding SP1 or NC to wild-type or mutant LPAR1 (transfected cells are HEK293). ( B ) Report of relative double luciferase after the 5-AZA treatment of HEK293 cells. ( C ) The specific binding of LPAR1 to SP1 was identified by Electrophoretic Mobility Shift Assay (EMSA). ( D ) The relative mRNA expression level of LPAR1 after the overexpression of SP1 and 5-AZA-induced demethylation in C2C12 cells. ( E ) The Western blot showing the protein expression levels of LPAR1 in C2C12 myoblasts after 5-aza-induced demethylation and SP1 overexpression. ( F ) Immunofluorescence detection of C2C12 myoblast differentiation after LPAR1 gene knockdown. The magnification of the picture is 10×, that is, the magnification of the objective lens is 10 times. ( G ) Relative mRNA expression level of myogenic marker genes after knockdown of the LPAR1 gene. ( H , I ) Relative mRNA expression levels of myogenic marker genes after SP1 gene knockdown and 5AZA-induced demethylation in C2C12 cells. The data are shown as the mean ± SD ( n = 3), *, p < 0.05 , **, p < 0.01 , ***, p < 0.001 .

Article Snippet: For the supershift assay, an anti-SP1 antibody (Santa Cruz biotechnology, Dallas, TX, USA) was added to the mixture and incubated on ice for 30 min. DNA–protein complexes were separated by non-denaturing 5% PAGE in 0.5× Tris-borate-EDTA (TBE) buffer, followed by electroblotting onto a positively charged nylon membrane.

Techniques: DNA Methylation Assay, Gene Expression, Binding Assay, Luciferase, Mutagenesis, Transfection, Electrophoretic Mobility Shift Assay, Expressing, Over Expression, Western Blot, Immunofluorescence, Knockdown, Marker