Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates expression of phenotypic switching markers of vascular smooth muscle cells ( VSMC s). A, The platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) decreases the expression levels of α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) at the protein level. PDGF ‐ BB does not affect the expression level of AGGF 1. B, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the protein level. C, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the mRNA level. NC indicates negative control. D, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in mouse VSMC line MOVAS ‐1 VSMC s. E, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in primary VSMC s isolated from mouse aortas. F, The expression levels of α‐ SMA , SM 22, and MYH 11 in MOVAS ‐1 VSMC s are significantly less than in primary mouse aortic VSMC s. G, Knockdown of SRF encoding the serum response factor by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the protein level. H, Knockdown of SRF by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the mRNA level. * P <0.05 (n=3/group). NS indicates not significant.

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Expressing, Derivative Assay, Negative Control, Isolation, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates transcriptional activation of vascular smooth muscle cells ( VSMC s) phenotypic switching markers. A, Luciferase assays showing that AGGF 1 increases transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) in the presence of SRF (serum response factor) ( SRF vs SRF +rh AGGF 1). NC indicates negative control. B, Luciferase assays showing that the platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) represses SRF ‐induced transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA , SM 22, and MYH 11, but the effects are abolished by AGGF 1 protein. C, Chromatin immunoprecipitation assays to detect protein– DNA interaction between SRF and the CA rG elements at the promoter/regulatory regions of VSMC s contractile marker genes. PDGF reduces the SRF binding to CA rG elements, but the effects are abolished by AGGF 1 protein. D, Co‐immunoprecipitation assays showing that the AGGF 1 protein increases the interaction between SRF and myocardin in MOVAS ‐1 VSMC s with overexpression of both myocardin and SRF . An anti‐myocardin antibody was used for immunoprecipitation, and an anti‐ SRF antibody was used for immunoblotting. E, Co‐immunoprecipitation assays showing that PDGF reduced the interaction between SRF and myocardin, but the effect was reversed by AGGF 1. * P <0.05 and ** P <0.01 (n=3/group).

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Activation Assay, Luciferase, Marker, Negative Control, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Over Expression, Western Blot

Journal: Nature Communications

Article Title: MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation

doi: 10.1038/s41467-019-09636-6

Figure Lengend Snippet: MKL1 activity results in reduced chromatin accessibility. a Quantification of the size distribution of DNase I digested DNA fragments from control iPS cells and caMKL1-blocked cells. n = 3 for each genotype. b FRAP analysis of the dynamic movement of histone H1 0 -mCherry in control iPS cell nuclei or caMKL1-blocked cell nuclei. **** P < 0.0001. n denotes the number of analyzed nuclei. Statistics were performed using two-tailed paired t -test. Error bars denote standard deviation. c , d Quantification of the size distribution of DNase I digested DNA fragments from WT and caMKL1-overexpressing ES cells ( c ) and SRF f/f iPS cells and SRF Δ/Δ iPS cells ( d ). n = 3 for each genotype. e , f MA plots of differential ATAC-seq peaks in WT vs caMKL1-expressing ES cells ( e ) and SRF f/f vs SRF Δ/Δ iPS cells ( f ). Pink dots indicate regions of significant differences in chromatin accessibility (left). The width of boxplots (right) is proportional to the number of regions of differential chromatin accessibility, showing overall lower accessibility in caMKL1-expressing ESCs ( e ), and overall increased accessibility in SRF Δ/Δ iPS cells ( f ). The center line of boxplots indicate the median value; the edges of the boxplots are at the 25th and 75th percentile; the whiskers extend to 1.5 times the interquartile range past the box. Points that are outside whiskers are shown individually. g Quantification of the size distribution of DNase I digested DNA fragments from caMKL1-blocked cells treated with or without UNC0642. n = 3 for each genotype. h Percentage of Oct4:GFP+ cells emerging from blocked cells following treatment with UNC0642. GFP+ cells were determined by FACS. All data shown are representative of three independent experiments

Article Snippet: For immunoprecipitation, Oct4 antibody (Cell Signaling, 5677S) and SRF antibody (Active Motif, 61385, Santa Cruz, sc-335) were used at 6 μg/ChIP sample, H3K4me3 antibody (Millipore, 07-473), H3K27ac antibody (Abcam, Ab4729), and H3K27me3 antibody (Millipore, 07-449) were used at 1 μg/ChIP sample.

Techniques: Activity Assay, Two Tailed Test, Standard Deviation, Expressing