Journal: The Journal of Biological Chemistry

Article Title: Foxa1 Functions as a Pioneer Transcription Factor at Transposable Elements to Activate Afp during Differentiation of Embryonic Stem Cells *

doi: 10.1074/jbc.M109.088096

Figure Lengend Snippet: Foxa1, Smad4, and TGF-β signaling are required for activation of Afp expression. A, RT-PCR was performed on RNA extracted from RA-treated ES cells transfected with Foxa1-targeted siRNA oligos, Smad4-target siRNA oligos, or scrambled siRNA oligos. Ct values from amplification of the indicated genes were normalized to 18S and graphed relative to the non-target siRNA. Error bars represent standard deviation from at least three repetitions. B, RT-PCR was performed on RNA extracted from Foxa1 null and wild type ES cells treated with RA for 0 or 4 days. Ct values from amplification of the indicated genes were normalized to 18S and graphed relative to the 0 day wild type sample. C, Western blots for P-Smad2 and β-actin were performed on protein lysates from ES cells treated with RA ± SB431542 for 4 days. D, RT-PCR was performed on RNA extracted from ES cells treated with RA ± SB431542 for 4 days. Expression of the indicated genes was normalized to 18S and graphed relative to the RA-treated sample.

Article Snippet: Primary antibodies for the following proteins were incubated overnight at 4 °C and used at the specified dilutions: H3K9ac (Upstate 06-942) 1:1000, H3K4me2 (Upstate 07-030) 1:2000, H3K4me3 (Novus NB 500-173) 1:2000, H3K9me2 (Abcam ab7312, Upstate 07-212) 1:1000, H3 (Abcam 1791) 1:5000, Foxa1 (Abcam ab5089) 1:500, Smad4 (Upstate DPC4) 1:500, β-actin (Genetex GTX30632, San Antonio, TX) 1:5000, Smad2 (Zymed Laboratories Inc. 51-1300) 1:1000, and phosphorylated Smad2 (P-Smad2) (Chemicon AB3849) 1:1000.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Amplification, Standard Deviation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Foxa1 Functions as a Pioneer Transcription Factor at Transposable Elements to Activate Afp during Differentiation of Embryonic Stem Cells *

doi: 10.1074/jbc.M109.088096

Figure Lengend Snippet: Activated Smad proteins bind chromatin at the Afp distal promoter only when Foxa1 is bound and silent chromatin is altered. A–C, E, and F, chromatin immunoprecipitation was performed on ES cells maintained in LIF or treated with RA for 4 days. DNA from immunoprecipitations for Foxa1 (A), Smad4 (B), P-Smad2 (C), H3 and H1 (F) was analyzed by real time PCR for binding to the Afp distal promoter and to the Nanog p53 response element. Results were graphed as percent bound minus IgG relative to input (A-C) or as normalized to LIF (F). E, re-ChIP analysis reveals simultaneous association of Foxa1 and Smad4 at the AFP distal promoter in RA-treated ES cells, but not in LIF-treated ES cells. A 1° ChIP was done with Smad4 and reactions were pooled, released from protein A beads, and then reimmunoprecipitated for IgG or Foxa1. Error bars represent S.D. of at least three repetitions; ns, not significant. D, whole cell lysates from ES cells maintained with LIF or differentiated with RA were probed with antibodies to Smad4, Smad2, P-Smad2, and β-actin. Triangles indicate differential loading.

Article Snippet: Primary antibodies for the following proteins were incubated overnight at 4 °C and used at the specified dilutions: H3K9ac (Upstate 06-942) 1:1000, H3K4me2 (Upstate 07-030) 1:2000, H3K4me3 (Novus NB 500-173) 1:2000, H3K9me2 (Abcam ab7312, Upstate 07-212) 1:1000, H3 (Abcam 1791) 1:5000, Foxa1 (Abcam ab5089) 1:500, Smad4 (Upstate DPC4) 1:500, β-actin (Genetex GTX30632, San Antonio, TX) 1:5000, Smad2 (Zymed Laboratories Inc. 51-1300) 1:1000, and phosphorylated Smad2 (P-Smad2) (Chemicon AB3849) 1:1000.

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Foxa1 Functions as a Pioneer Transcription Factor at Transposable Elements to Activate Afp during Differentiation of Embryonic Stem Cells *

doi: 10.1074/jbc.M109.088096

Figure Lengend Snippet: Foxa1 acts upstream of Smads to mediate Smad binding, nucleosome occupancy reduction, and H3K9 acetylation. Chromatin immunoprecipitation was performed on ES cells treated with RA for 4 days and transfected with either Foxa1-targeted or scrambled siRNA oligos (A, C, and H) or treated with SB431542 or dimethyl sulfoxide (B, F, and I). Transfection of the siRNA oligos for Foxa1 or for a non-target control and treatment with SB431542 or dimethyl sulfoxide occurred coincident with addition of RA. DNA from immunoprecipitations for H3 (A and B), Foxa1, P-Smad2, and Smad4 (C, D, and F), and H3 and H3K9ac (E and G–I) was analyzed by real time PCR for binding to the Afp distal promoter. DNA from immunoprecipitations was analyzed by real time PCR for binding to the Afp distal promoter. Levels of acH3K9 are expressed as a ratio to levels of histone H3, determined by a separate immunoprecipitation. Error bars represent S.D. from at least three repetitions. ns, not significant.

Article Snippet: Primary antibodies for the following proteins were incubated overnight at 4 °C and used at the specified dilutions: H3K9ac (Upstate 06-942) 1:1000, H3K4me2 (Upstate 07-030) 1:2000, H3K4me3 (Novus NB 500-173) 1:2000, H3K9me2 (Abcam ab7312, Upstate 07-212) 1:1000, H3 (Abcam 1791) 1:5000, Foxa1 (Abcam ab5089) 1:500, Smad4 (Upstate DPC4) 1:500, β-actin (Genetex GTX30632, San Antonio, TX) 1:5000, Smad2 (Zymed Laboratories Inc. 51-1300) 1:1000, and phosphorylated Smad2 (P-Smad2) (Chemicon AB3849) 1:1000.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Journal of the American Heart Association

Article Title: Deletion of Microfibrillar‐Associated Protein 4 Attenuates Left Ventricular Remodeling and Dysfunction in Heart Failure

doi: 10.1161/jaha.119.015307

Figure Lengend Snippet: Figure 4. MFAP4 deficiency attenuates aortic banding-induced cardiac fibrosis. A, Representative image of the heart with Picrosirius staining. scale bar: 50 μm. B, Quantification of the total collagen volume in the indicated group (n=6). C through H, PCR analysis of fibrotic markers (αSMA, collagen Iα, collagen III, CTGF, fibronectin, TGF-β) (n=6). I through M, Representative blots and quantitative results for αSMA, collagen III and P-Smad2/T-Smad2, and P-Smad3/T-Smad3 protein expression in the myocardium in each group (n=6). AB indicates aortic banding; Col1α, collagen Iα; Col3, collagen type 3; CTGF, connective tissue growth factor; KO, knockout mice; MFAP4, microfibrillar-associated protein 4; PSR, Picrosirius red; TGF-β, transforming growth factor-β; WT, wild-type; and αSMA, alpha-smooth muscle actin. *P<0.05 compared with the corresponding wild- type-aortic banding group.

Article Snippet: The following main antibodies were obtained from Abcam (Cambridge, UK): inducible MFAP4 (#ab 80319), transforming growth factor-β (TGFβ, #ab64715), α-smooth muscle actin (α-SMA, #ab5694), Connexin 43 (Cx43, #ab11370); The antibodies against GAPDH (#2118), focal adhesion kinase (FAK, #3285), P-FAK (#3283), phosphorylated-Smad2 (P-Smad2, #3108s), P-Smad3 (#8769), total-Smad2 (T-Smad2, #3103s), T-Smad3 (# 9513s), total—extracellular regulated protein kinase 1/2 (T-ERK1/2, #4695), P-ERK1/2 (#4370P), T-AKT (4691#), P-AKT (4060#), T-PI3K (#4257), P-PI3K (#4228S), T-MEK1/2 (#9122s), P-MEK1/2 (#9154s) were purchased from Cell Signaling Technology (Danvers, MA, USA); The antibodies against ANP (atrial natriuretic peptide) (# sc-20158), β-MHC (β-myosin heavy chain) (sc-53090), Col2a1 (collagen type III alpha 1) (sc-8781) were obtained from Santa Cruz Inc. (TX, USA).

Techniques: Staining, Expressing, Knock-Out