anti-srf antibody Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Millipore anti srf antibody
    Anti Srf Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf antibody/product/Millipore
    Average 93 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    anti srf antibody - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology anti srf antibody
    Micro-topographies elevate the early response genes <t>SRF</t> and <t>EGR1</t> during the cells early adaptation phase. A) In silico design of the PS-1018 surface used in subsequent experiments. Scale bar represent 10 μm. B) Similar as the PS-281 surface, the PS-1018 surface elicits smaller, elongated morphological characteristics and a reduction in F-actin stress fibers. F-actin was immunolabeled by phalloidin (yellow) and the nucleus counterstained with Hoechst33342 (magenta). Scale bar represent 50 μm. C) Immunolabeling of F-actin after 1h of cell culture reveals that MSCs on a flat surface exhibit a round morphology with a diffuse F-actin pattern. D-E) Immunolabeling of F-actin of MSCs cultured for 1h on the PS-1018 surface reveals an increase in F-actin stress fibers concentrated on the upper part of the micro-topographical structures, while a diffuse pattern is observed at the bottom of the PS-1018 structures. Scale bar represent 10 μm. F) EGR1 levels are elevated at 24h as measured through qPCR (* P
    Anti Srf Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf antibody/product/Santa Cruz Biotechnology
    Average 89 stars, based on 189 article reviews
    Price from $9.99 to $1999.99
    anti srf antibody - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    89
    Cell Signaling Technology Inc anti srf antibody
    <t>SRF</t> is critical for <t>YAP</t> induction of MaSC-like properties. ( a ) Summary of GSEA analysis for sets of transcription factor targets. The top 10 gene sets enriched in MCF-10A ER T2 -YAP 2SA cells and top three gene sets enriched in MCF-10A ER T2 cells with lowest false discovery rate are shown. Full GSEA analysis results are presented in Supplementary Data 2 . ( b ) qRT–PCR analyses of YAP-overexpressing MCF-10A cells infected with shRNA targeting transcription factors or coexpressing the dominant-negative transcription factor LIP ( n =2 replicates). Refer to Supplementary Fig. 7b for successful expression of shRNA or dominant-negative transcription factor. ( c , d ) Distinct transcriptional regulation of YAP target genes by SRF and TEAD. qRT–PCR analyses of MCF-10A cells overexpressing YAP and shRNA against ( c ) SRF ( n =2 replicates) or ( d ) TEAD1/3/4 ( n =2 replicates). ( e , f ) SRF is required for YAP induction of MaSC-like properties. ( e ) Representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( f ) the number of mammospheres formed by MCF-10A cells expressing YAP and shRNA against SRF ( n =3 experiments). Data are presented as means±s.e.m. (* P
    Anti Srf Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf antibody/product/Cell Signaling Technology Inc
    Average 89 stars, based on 49 article reviews
    Price from $9.99 to $1999.99
    anti srf antibody - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc anti phospho srf
    <t>SRF</t> is critical for <t>YAP</t> induction of MaSC-like properties. ( a ) Summary of GSEA analysis for sets of transcription factor targets. The top 10 gene sets enriched in MCF-10A ER T2 -YAP 2SA cells and top three gene sets enriched in MCF-10A ER T2 cells with lowest false discovery rate are shown. Full GSEA analysis results are presented in Supplementary Data 2 . ( b ) qRT–PCR analyses of YAP-overexpressing MCF-10A cells infected with shRNA targeting transcription factors or coexpressing the dominant-negative transcription factor LIP ( n =2 replicates). Refer to Supplementary Fig. 7b for successful expression of shRNA or dominant-negative transcription factor. ( c , d ) Distinct transcriptional regulation of YAP target genes by SRF and TEAD. qRT–PCR analyses of MCF-10A cells overexpressing YAP and shRNA against ( c ) SRF ( n =2 replicates) or ( d ) TEAD1/3/4 ( n =2 replicates). ( e , f ) SRF is required for YAP induction of MaSC-like properties. ( e ) Representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( f ) the number of mammospheres formed by MCF-10A cells expressing YAP and shRNA against SRF ( n =3 experiments). Data are presented as means±s.e.m. (* P
    Anti Phospho Srf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho srf/product/Cell Signaling Technology Inc
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    anti phospho srf - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    91
    Millipore mouse anti srf
    The transcription factors Elk-1 and <t>SRF</t> bind to the <t>Mcl-1</t> promoter in MCF-7 cells. ( a ) Chromatin immunoprecipitation using antibodies specific to Elk-1 and SRF was performed as described in Material and methods section. Controls shown are two negative antibody controls as well as a no antibody control (protein G beads alone). Ctrl1 is an antibody against Stat-3 and Ctrl 2 is an antibody against NF-κB. Fold enrichment values were obtained by comparing the cT values for each ChIP sample to an equal amount of input DNA. Primers were designed in the last exon of the Mcl-1 gene to demonstrate specificity (RT–PCR data not shown, PCR products shown in ( b )). Data represents the mean of three independent experiments±standard error. ( b ) PCR products were run on an agarose gel following each ChIP experiment to assess reaction specificity. ( c ) Streptavidin pull-down assay to detect transcription factor binding to a 50 bp double-stranded biotin labelled probe specific to the Mcl-1 promoter region of interest. Cells were stimulated with EGF for different time periods and nuclear extracts incubated with the Mcl-1 probe. Following pull down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labelled scrambled probe was used along with competition with an unlabelled specific probe. Blots were probed with antibodies to the transcription factors Stat-3 and NF-κB to demonstrate binding specificity. ( d ) Schematic representation of the Mcl-1 gene showing approximate locations of the biotin labelled probe used for the Streptavidin pull down as well as the primers used for ChIP.
    Mouse Anti Srf, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti srf/product/Millipore
    Average 91 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse anti srf - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology anti srf monoclonal antibody
    The transcription factors Elk-1 and <t>SRF</t> bind to the <t>Mcl-1</t> promoter in MCF-7 cells. ( a ) Chromatin immunoprecipitation using antibodies specific to Elk-1 and SRF was performed as described in Material and methods section. Controls shown are two negative antibody controls as well as a no antibody control (protein G beads alone). Ctrl1 is an antibody against Stat-3 and Ctrl 2 is an antibody against NF-κB. Fold enrichment values were obtained by comparing the cT values for each ChIP sample to an equal amount of input DNA. Primers were designed in the last exon of the Mcl-1 gene to demonstrate specificity (RT–PCR data not shown, PCR products shown in ( b )). Data represents the mean of three independent experiments±standard error. ( b ) PCR products were run on an agarose gel following each ChIP experiment to assess reaction specificity. ( c ) Streptavidin pull-down assay to detect transcription factor binding to a 50 bp double-stranded biotin labelled probe specific to the Mcl-1 promoter region of interest. Cells were stimulated with EGF for different time periods and nuclear extracts incubated with the Mcl-1 probe. Following pull down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labelled scrambled probe was used along with competition with an unlabelled specific probe. Blots were probed with antibodies to the transcription factors Stat-3 and NF-κB to demonstrate binding specificity. ( d ) Schematic representation of the Mcl-1 gene showing approximate locations of the biotin labelled probe used for the Streptavidin pull down as well as the primers used for ChIP.
    Anti Srf Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf monoclonal antibody/product/Santa Cruz Biotechnology
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    anti srf monoclonal antibody - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology rabbit anti srf antibodies
    Actin signaling modulates neuronal gene expression. A , R62D decreased, G15S increased, and S14C did not influence <t>SRF</t> luciferase activity in neurons. B , R62D antagonized constitutively active SRF (SRF-VP16)-induced luciferase activity. C–F , SRF-VP16 ( E ) partially rescued R62D-decreased neurite length in contrast to SRF-VP16ΔMADS ( D ) used as control (quantified in F ). G–M , R62D expression, confined to the nucleus (R62D NLS ), was recognized by its FLAG tag. R62D NLS -positive neurons ( H , J , L , arrows) showed severe structural abnormalities including decreased neurite length and lack of growth-cone filopodia. In control neurons ( K ), growth cones were collapsed by ephrin-A5 ( K , arrowheads). Contrastingly, ephrin-A5 failed to collapse a growth cone ( L , arrowhead) of an R62D NLS -expressing neuron ( L , arrow; quantified in M ). N , R62D NLS decreased SRF luciferase activity and counteracted SRF-VP16 enhanced gene activity (see B ). O , SRF-VP16 fully rescued R62D NLS -decreased neurite length. P , Cytoplasmic R62D and R62D NLS modulated expression of <t>MAL,</t> but not SRF. R62D NLS decreased expression of a 100 kDa MAL protein, but upregulated a MAL variant of ∼70 kDa. Cytoplasmic R62D increased abundance of the 70 kDa MAL protein, but not of 100 kDa MAL. Triplicate culture of three different mice for R62D and R62D NLS are shown. Scale bars ( C–E , G–L ), 5 μm.
    Rabbit Anti Srf Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti srf antibodies/product/Santa Cruz Biotechnology
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit anti srf antibodies - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Millipore mouse monoclonal anti srf antibody
    Serum response factor <t>(SRF)</t> and NK3 transcription factors are required for nuclear import in smooth muscle cells (SMCs). ( a ) Immunoblots of human pulmonary artery SMCs (hSMCs) transfected with nontargeting (NT) siRNA or siRNA targeting SRF, <t>NKX3-1</t> and
    Mouse Monoclonal Anti Srf Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti srf antibody/product/Millipore
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti srf antibody - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    86
    Abcam phospho srf
    Engineered transcription factors targeting the FosB promoter bidirectionally regulate gene expression in NAc neurons via epigenetic manipulation. (a) Locations of FosB -ZFP binding relative to the FosB TSS. The location of functional <t>SRF</t> and <t>CREB</t> binding
    Phospho Srf, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho srf/product/Abcam
    Average 86 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    phospho srf - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    85
    Upstate Biotechnology Inc anti srf antibodies
    Increased responsiveness to <t>SRF</t> in the intron 14 from SHR. (A) Analysis of intron 14 in other normotensive and hypertensive rat strains. Stroke-prone SHR (SHRSP) also contains the 12-base pair insertion, whereas normotensive SD and <t>WKY</t> rats do not. (B) Activity of intron 14 from WKY (WKY-pMK) rats, SD (SD-pMK) rats, and SHR (SHR-pMK) was analyzed using luciferase reporter gene assays. Although coexpression with 50 ng of the SRF plasmid increased the activities of all three promoters, it increased SHR promoter activity much more than the activities of the WKY and SD promoters (7.2-, 2.7-, and 2.1-fold compared with control cells transfected with 50 ng of pcDNA 3.1, respectively). The means ± SE are shown. *p
    Anti Srf Antibodies, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf antibodies/product/Upstate Biotechnology Inc
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    anti srf antibodies - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    93
    Abcam anti serum response factor srf antibody
    Co-expression of <t>αSMA</t> and <t>SRF</t> in part of stromal cells originated from bone marrow-derived cells. ( A ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed αSMA (indicated by arrows) in a carcinogen-induced small intestinal tumor. ( B ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed SRF (indicated by arrows) in the small intestinal tumor. A SRF(+) cell without GFP expression is indicated by a filled arrow.
    Anti Serum Response Factor Srf Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti serum response factor srf antibody/product/Abcam
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    anti serum response factor srf antibody - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology anti srf polyclonal rabbit antibody
    Co-expression of <t>αSMA</t> and <t>SRF</t> in part of stromal cells originated from bone marrow-derived cells. ( A ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed αSMA (indicated by arrows) in a carcinogen-induced small intestinal tumor. ( B ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed SRF (indicated by arrows) in the small intestinal tumor. A SRF(+) cell without GFP expression is indicated by a filled arrow.
    Anti Srf Polyclonal Rabbit Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf polyclonal rabbit antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    anti srf polyclonal rabbit antibody - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    91
    Active Motif anti srf antibody
    <t>mDia2-SRF</t> signaling is involved in the regulation of HSPC engraftment. a Immunofluorescence staining of HSPCs with F-actin (red) and α-tubulin. White arrows: F-actin protrusions. Yellow arrows: polarization axis to determine the staining intensity in b . The experiments were repeated three times with similar results. b F-actin and α-tubulin staining intensity across the yellow arrows from a . c Quantitative analyses of F-actin staining intensity from a . N = 17 cells in mDia2 fl/fl group. N = 13 cells in mDia2 fl/fl Vav-Cre group. d Quantitative analyses of the percentage of cells with F-actin protrusions in a . N = 5 fields in mDia2 fl/fl group. N = 6 fields in mDia2 fl/fl Vav-Cre group. e Quantitative analysis of the percentages of <t>MAL</t> nuclear localization in c-Kit+ HSPCs incubated with or without FBS for 15 min. mDia2 fl/fl no FBS: N = 71 cells from 17 random fields; mDia2 fl/fl Vav-Cre no FBS: N = 80 cells from 15 random fields; mDia2 fl/fl with 10% FBS: N = 56 cells from 12 random fields; mDia2 fl/fl Vav-Cre with 10% FBS: N = 108 cells from 19 random fields. Data are from three independent experiments. f Quantitative RT-PCR of the indicated SRF target genes in c-Kit+ HSPCs from the indicated mice. g Quantitative RT-PCR of the indicated integrins in c-Kit+ HSPCs from the indicated mice. The experiments were performed in triplicate from n = 5 mice per group in f , g . h Quantitative analyses of the surface expression level of beta2 integrins using flow cytometry in the indicated HSPC populations. n = 2 mice per group. i Representative flow cytometry plots showing the expression of beta2 integrins in ex vivo-cultured c-Kit+ HSPCs. j Quantitative analyses of i . n = 2 mice per group. k In vitro adhesion assay of the cultured c-kit+ HSPCs from the indicated mice on extracellular matrix protein coated coverslips. Data are presented as adhesion ability relative to the control cells. mDia2 fl/fl , Collagen: 25 fields, Fibronectin: 20 fields, poly- d -lysine: 15 fields. mDia2 fl/fl Vav-Cre, Collagen: 10 fields, Fibronectin: 14 fields, poly- d -lysine: 9 fields. l In vitro adhesion assay of indicated c-kit+ HSPCs cultured on ICAM-1-coated coverslips. Error bars represent the SEM of the mean. * p
    Anti Srf Antibody, supplied by Active Motif, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti srf antibody/product/Active Motif
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti srf antibody - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology a11 anti srf antibody
    <t>mDia2-SRF</t> signaling is involved in the regulation of HSPC engraftment. a Immunofluorescence staining of HSPCs with F-actin (red) and α-tubulin. White arrows: F-actin protrusions. Yellow arrows: polarization axis to determine the staining intensity in b . The experiments were repeated three times with similar results. b F-actin and α-tubulin staining intensity across the yellow arrows from a . c Quantitative analyses of F-actin staining intensity from a . N = 17 cells in mDia2 fl/fl group. N = 13 cells in mDia2 fl/fl Vav-Cre group. d Quantitative analyses of the percentage of cells with F-actin protrusions in a . N = 5 fields in mDia2 fl/fl group. N = 6 fields in mDia2 fl/fl Vav-Cre group. e Quantitative analysis of the percentages of <t>MAL</t> nuclear localization in c-Kit+ HSPCs incubated with or without FBS for 15 min. mDia2 fl/fl no FBS: N = 71 cells from 17 random fields; mDia2 fl/fl Vav-Cre no FBS: N = 80 cells from 15 random fields; mDia2 fl/fl with 10% FBS: N = 56 cells from 12 random fields; mDia2 fl/fl Vav-Cre with 10% FBS: N = 108 cells from 19 random fields. Data are from three independent experiments. f Quantitative RT-PCR of the indicated SRF target genes in c-Kit+ HSPCs from the indicated mice. g Quantitative RT-PCR of the indicated integrins in c-Kit+ HSPCs from the indicated mice. The experiments were performed in triplicate from n = 5 mice per group in f , g . h Quantitative analyses of the surface expression level of beta2 integrins using flow cytometry in the indicated HSPC populations. n = 2 mice per group. i Representative flow cytometry plots showing the expression of beta2 integrins in ex vivo-cultured c-Kit+ HSPCs. j Quantitative analyses of i . n = 2 mice per group. k In vitro adhesion assay of the cultured c-kit+ HSPCs from the indicated mice on extracellular matrix protein coated coverslips. Data are presented as adhesion ability relative to the control cells. mDia2 fl/fl , Collagen: 25 fields, Fibronectin: 20 fields, poly- d -lysine: 15 fields. mDia2 fl/fl Vav-Cre, Collagen: 10 fields, Fibronectin: 14 fields, poly- d -lysine: 9 fields. l In vitro adhesion assay of indicated c-kit+ HSPCs cultured on ICAM-1-coated coverslips. Error bars represent the SEM of the mean. * p
    A11 Anti Srf Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a11 anti srf antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    a11 anti srf antibody - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology polyclonal anti srf antibody
    (A) Effect of PIAS1 on the formation of <t>CArG-SRF</t> complex containing myocardin by EMSA. Rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and nuclear extracts were prepared and incubated with a 32 P labeled 95-bp probe corresponding to the −137 to −42 bp region of the SM α-actin gene. Nuclear extracts from bovine aortic EC were used as a control. Arrowheads A and B indicate the DNA protein complex from EC and SMC, respectively. <t>Polyclonal</t> antibody against SRF or control IgG was added for supershift. (B) ChIP assays. Cultured rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and SRF binding to the CArG region within intact chromatin was examined by ChIP assays as described in Materials and Methods. Quantitative PCR was used to detect CArG-containing regions of the SM α-actin promoter in chromatin fragments immunoprecipitated with anti-SRF antibody. An n -fold enrichment of 100 was assigned to SRF association in cells overexpressing control (αGFP). Values present the means ± standard errors of the mean. *, P of
    Polyclonal Anti Srf Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti srf antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti srf antibody - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    Micro-topographies elevate the early response genes SRF and EGR1 during the cells early adaptation phase. A) In silico design of the PS-1018 surface used in subsequent experiments. Scale bar represent 10 μm. B) Similar as the PS-281 surface, the PS-1018 surface elicits smaller, elongated morphological characteristics and a reduction in F-actin stress fibers. F-actin was immunolabeled by phalloidin (yellow) and the nucleus counterstained with Hoechst33342 (magenta). Scale bar represent 50 μm. C) Immunolabeling of F-actin after 1h of cell culture reveals that MSCs on a flat surface exhibit a round morphology with a diffuse F-actin pattern. D-E) Immunolabeling of F-actin of MSCs cultured for 1h on the PS-1018 surface reveals an increase in F-actin stress fibers concentrated on the upper part of the micro-topographical structures, while a diffuse pattern is observed at the bottom of the PS-1018 structures. Scale bar represent 10 μm. F) EGR1 levels are elevated at 24h as measured through qPCR (* P

    Journal: bioRxiv

    Article Title: Surface topography is a context-dependent activator of TGF-β signaling in mesenchymal stem cells

    doi: 10.1101/2020.01.13.903195

    Figure Lengend Snippet: Micro-topographies elevate the early response genes SRF and EGR1 during the cells early adaptation phase. A) In silico design of the PS-1018 surface used in subsequent experiments. Scale bar represent 10 μm. B) Similar as the PS-281 surface, the PS-1018 surface elicits smaller, elongated morphological characteristics and a reduction in F-actin stress fibers. F-actin was immunolabeled by phalloidin (yellow) and the nucleus counterstained with Hoechst33342 (magenta). Scale bar represent 50 μm. C) Immunolabeling of F-actin after 1h of cell culture reveals that MSCs on a flat surface exhibit a round morphology with a diffuse F-actin pattern. D-E) Immunolabeling of F-actin of MSCs cultured for 1h on the PS-1018 surface reveals an increase in F-actin stress fibers concentrated on the upper part of the micro-topographical structures, while a diffuse pattern is observed at the bottom of the PS-1018 structures. Scale bar represent 10 μm. F) EGR1 levels are elevated at 24h as measured through qPCR (* P

    Article Snippet: Primary antibodies used in this study are: anti-SCX antibody (1:200; Abcam; ab58655), anti-EGR1 antibody (1:200; ThermoFisher; T.126.1), anti-Phospho-Smad2/3 (1:200; Cell Signaling Technologies; 8828S) and anti-SRF antibody (1:200; Santa Cruz; sc-335).

    Techniques: In Silico, Immunolabeling, Cell Culture, Real-time Polymerase Chain Reaction

    SRF is critical for YAP induction of MaSC-like properties. ( a ) Summary of GSEA analysis for sets of transcription factor targets. The top 10 gene sets enriched in MCF-10A ER T2 -YAP 2SA cells and top three gene sets enriched in MCF-10A ER T2 cells with lowest false discovery rate are shown. Full GSEA analysis results are presented in Supplementary Data 2 . ( b ) qRT–PCR analyses of YAP-overexpressing MCF-10A cells infected with shRNA targeting transcription factors or coexpressing the dominant-negative transcription factor LIP ( n =2 replicates). Refer to Supplementary Fig. 7b for successful expression of shRNA or dominant-negative transcription factor. ( c , d ) Distinct transcriptional regulation of YAP target genes by SRF and TEAD. qRT–PCR analyses of MCF-10A cells overexpressing YAP and shRNA against ( c ) SRF ( n =2 replicates) or ( d ) TEAD1/3/4 ( n =2 replicates). ( e , f ) SRF is required for YAP induction of MaSC-like properties. ( e ) Representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( f ) the number of mammospheres formed by MCF-10A cells expressing YAP and shRNA against SRF ( n =3 experiments). Data are presented as means±s.e.m. (* P

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: SRF is critical for YAP induction of MaSC-like properties. ( a ) Summary of GSEA analysis for sets of transcription factor targets. The top 10 gene sets enriched in MCF-10A ER T2 -YAP 2SA cells and top three gene sets enriched in MCF-10A ER T2 cells with lowest false discovery rate are shown. Full GSEA analysis results are presented in Supplementary Data 2 . ( b ) qRT–PCR analyses of YAP-overexpressing MCF-10A cells infected with shRNA targeting transcription factors or coexpressing the dominant-negative transcription factor LIP ( n =2 replicates). Refer to Supplementary Fig. 7b for successful expression of shRNA or dominant-negative transcription factor. ( c , d ) Distinct transcriptional regulation of YAP target genes by SRF and TEAD. qRT–PCR analyses of MCF-10A cells overexpressing YAP and shRNA against ( c ) SRF ( n =2 replicates) or ( d ) TEAD1/3/4 ( n =2 replicates). ( e , f ) SRF is required for YAP induction of MaSC-like properties. ( e ) Representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( f ) the number of mammospheres formed by MCF-10A cells expressing YAP and shRNA against SRF ( n =3 experiments). Data are presented as means±s.e.m. (* P

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

    Techniques: Quantitative RT-PCR, Infection, shRNA, Dominant Negative Mutation, Expressing, FACS

    SRF–YAP–IL6 signalling is upregulated in basal-type breast cancer. ( a , b ) SRF–YAP/TAZ–IL6 are highly expressed BLBC. ( a ) Heatmap analysis ( b ) and statistical analysis of the expression of the indicated genes in a panel of breast cancers (GSE31448; n > 27 for each subtype). ( c , d ) A tissue microarray of breast cancers was stained with antibodies against the indicated proteins. ( c ) Representative immunohistochemical data for different types of breast cancers. Scale bar, 100 μm. ( d ) Summary and statistical analysis of tissue microarray data. P values were calculated using Fisher's exact test. ( e ) Scatter plot of signature score for the indicated cell types against YAP+TAZ or SRF expression levels ( n =357; correlation tested using Pearson's correlation coefficient, r ). ( f ) Scatter plot of IL6 and CTGF expression against YAP+TAZ or SRF expression levels ( n =357; correlation tested using Pearson's correlation coefficient, r ). Data are presented as means±s.e.m. (** P

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: SRF–YAP–IL6 signalling is upregulated in basal-type breast cancer. ( a , b ) SRF–YAP/TAZ–IL6 are highly expressed BLBC. ( a ) Heatmap analysis ( b ) and statistical analysis of the expression of the indicated genes in a panel of breast cancers (GSE31448; n > 27 for each subtype). ( c , d ) A tissue microarray of breast cancers was stained with antibodies against the indicated proteins. ( c ) Representative immunohistochemical data for different types of breast cancers. Scale bar, 100 μm. ( d ) Summary and statistical analysis of tissue microarray data. P values were calculated using Fisher's exact test. ( e ) Scatter plot of signature score for the indicated cell types against YAP+TAZ or SRF expression levels ( n =357; correlation tested using Pearson's correlation coefficient, r ). ( f ) Scatter plot of IL6 and CTGF expression against YAP+TAZ or SRF expression levels ( n =357; correlation tested using Pearson's correlation coefficient, r ). Data are presented as means±s.e.m. (** P

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

    Techniques: Expressing, Microarray, Staining, Immunohistochemistry

    Interaction with SRF recruits YAP to MaSC signature-gene promoters. ( a ) ChIP–qPCR analyses of the indicated transcriptional regulators ( n =3 experiments). ( b ) Luciferase assay of the IL6 promoter CArG box ( n =3 experiments). ( c ) EMSA assay of the IL6 promoter CArG box using 293T extracts expressing indicated genes and GST-tagged proteins. ( d ) ChIP–qPCR analyses of the indicated cells ( n =3 experiments). ( e ) ChIP–qPCR analyses of the TEAD-binding boxes of CTGF/CYR61 promoters and CArG boxes of IL6/DLL1 promoters in MCF-10A cells expressing the indicated mutant YAP ( n =3 experiments). YAP 5SA mutants are used to maximize chromatin binding. Data are presented as means±s.e.m. (* P

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: Interaction with SRF recruits YAP to MaSC signature-gene promoters. ( a ) ChIP–qPCR analyses of the indicated transcriptional regulators ( n =3 experiments). ( b ) Luciferase assay of the IL6 promoter CArG box ( n =3 experiments). ( c ) EMSA assay of the IL6 promoter CArG box using 293T extracts expressing indicated genes and GST-tagged proteins. ( d ) ChIP–qPCR analyses of the indicated cells ( n =3 experiments). ( e ) ChIP–qPCR analyses of the TEAD-binding boxes of CTGF/CYR61 promoters and CArG boxes of IL6/DLL1 promoters in MCF-10A cells expressing the indicated mutant YAP ( n =3 experiments). YAP 5SA mutants are used to maximize chromatin binding. Data are presented as means±s.e.m. (* P

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

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

    SRF interacts with YAP. ( a ) Western blot and qRT–PCR analyses ( n =2 replicates) of MCF-10A cells coexpressing YAP and TEAD2/SRF. Note that SRF, but not TEAD2, synergized with YAP in promoting IL6 expression. ( b ) Western blot showing YAP–SRF co-immunoprecipitation with SRF- and/or YAP-overexpressing 293T cell extract crosslinked with dithiobis(succinimidyl propionate) (DSP). ( c , d ) Western blot showing endogenous YAP–SRF co-immunoprecipitation with ( c ) YAP antibody or ( d ) SRF antibody in 293T cell extract crosslinked with DSP. ( e ) Western blot showing co-immunoprecipitation with the indicated Flag-tagged mutant YAP and SRF. Note that YAP S94A fails to bind SRF. ( f ) In vitro pulldown assay with purified S-tag-Flag-SRF and bacterially purified GST-tagged YAP mutants. Asterisk indicates nonspecific bands, and the arrow indicates the desired band.

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: SRF interacts with YAP. ( a ) Western blot and qRT–PCR analyses ( n =2 replicates) of MCF-10A cells coexpressing YAP and TEAD2/SRF. Note that SRF, but not TEAD2, synergized with YAP in promoting IL6 expression. ( b ) Western blot showing YAP–SRF co-immunoprecipitation with SRF- and/or YAP-overexpressing 293T cell extract crosslinked with dithiobis(succinimidyl propionate) (DSP). ( c , d ) Western blot showing endogenous YAP–SRF co-immunoprecipitation with ( c ) YAP antibody or ( d ) SRF antibody in 293T cell extract crosslinked with DSP. ( e ) Western blot showing co-immunoprecipitation with the indicated Flag-tagged mutant YAP and SRF. Note that YAP S94A fails to bind SRF. ( f ) In vitro pulldown assay with purified S-tag-Flag-SRF and bacterially purified GST-tagged YAP mutants. Asterisk indicates nonspecific bands, and the arrow indicates the desired band.

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Immunoprecipitation, Mutagenesis, In Vitro, Purification

    SRF–YAP–IL6 signalling is required for CSC formation. ( a – c ) YAP/TAZ knockdown decreases IL6 expression and CSC formation. MCF-10A cells were transformed with a constitutively active form of H-Ras (HRas G12V ) and then transduced with shRNA against the indicated genes. ( a ) Western blot and qRT–PCR analyses ( n =2 replicates), ( b ) representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( c ) statistical analyses of the number of mammospheres formed by MCF-10A cells expressing HRasG12V together with shRNA against YAP and/or TAZ ( n =3 experiments). ( d , e ) MCF-10A H-Ras G12V cells were infected with shRNA against SRF. ( d ) Western blot and qRT–PCR analyses ( n =2 replicates) and ( e ) statistical analyses of the number of mammospheres formed by cells infected with the indicated viruses ( n =3 experiments). ( f , g ) MCF-10A H-Ras G12V cells infected with shRNA against YAP/TAZ or SRF were ( f ) infected with IL6-expressing retrovirus or ( g ) were treated with recombinant IL6 (r-hIL6; 5 ng ml −1 ). Statistical analyses of the number of mammospheres formed by cells infected with the indicated viruses ( n =3 experiments). ( h ) Limiting dilution xenograft assays of cells generated in f . Data are presented as means±s.e.m. (* P

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: SRF–YAP–IL6 signalling is required for CSC formation. ( a – c ) YAP/TAZ knockdown decreases IL6 expression and CSC formation. MCF-10A cells were transformed with a constitutively active form of H-Ras (HRas G12V ) and then transduced with shRNA against the indicated genes. ( a ) Western blot and qRT–PCR analyses ( n =2 replicates), ( b ) representative FACS plot and statistical analyses of CD44 and CD24 antigen expression ( n =3 experiments) and ( c ) statistical analyses of the number of mammospheres formed by MCF-10A cells expressing HRasG12V together with shRNA against YAP and/or TAZ ( n =3 experiments). ( d , e ) MCF-10A H-Ras G12V cells were infected with shRNA against SRF. ( d ) Western blot and qRT–PCR analyses ( n =2 replicates) and ( e ) statistical analyses of the number of mammospheres formed by cells infected with the indicated viruses ( n =3 experiments). ( f , g ) MCF-10A H-Ras G12V cells infected with shRNA against YAP/TAZ or SRF were ( f ) infected with IL6-expressing retrovirus or ( g ) were treated with recombinant IL6 (r-hIL6; 5 ng ml −1 ). Statistical analyses of the number of mammospheres formed by cells infected with the indicated viruses ( n =3 experiments). ( h ) Limiting dilution xenograft assays of cells generated in f . Data are presented as means±s.e.m. (* P

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

    Techniques: Expressing, Transformation Assay, Transduction, shRNA, Western Blot, Quantitative RT-PCR, FACS, Infection, Recombinant, Generated

    SRF–YAP–IL6 signalling promotes cancer stemness in vivo . ( a ) Immunohistochemical analysis of serial sections of 4T1 syngeneic graft tumours. Scale bar, 100 μm. ( b , c ) 4T1 cells were infected with retrovirus overexpressing YAP and lentivirus expressing shRNA against the indicated genes. ( b ) qRT–PCR analysis of IL6 expression ( n =2 replicates) and ( c ) quantification of the number of mammospheres ( n =3 experiments). ( d ) ChIP–qPCR analyses of 4T1 cells infected with the indicated viruses ( n =3 experiments). ( e , f ) Cells generated in b were injected into BALB/c mice. ( e ) Representative tumour image. Blue asterisks indicate tumours. ( f ) Summary data of tumour-initiation frequency. Data are presented as means±s.e.m. (* P

    Journal: Nature Communications

    Article Title: A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    doi: 10.1038/ncomms10186

    Figure Lengend Snippet: SRF–YAP–IL6 signalling promotes cancer stemness in vivo . ( a ) Immunohistochemical analysis of serial sections of 4T1 syngeneic graft tumours. Scale bar, 100 μm. ( b , c ) 4T1 cells were infected with retrovirus overexpressing YAP and lentivirus expressing shRNA against the indicated genes. ( b ) qRT–PCR analysis of IL6 expression ( n =2 replicates) and ( c ) quantification of the number of mammospheres ( n =3 experiments). ( d ) ChIP–qPCR analyses of 4T1 cells infected with the indicated viruses ( n =3 experiments). ( e , f ) Cells generated in b were injected into BALB/c mice. ( e ) Representative tumour image. Blue asterisks indicate tumours. ( f ) Summary data of tumour-initiation frequency. Data are presented as means±s.e.m. (* P

    Article Snippet: ChIP assay DNA in cells from two confluent 100-mm culture dishes (∼2 × 107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 μg of anti-YAP antibody (H-125, Santa Cruz Biotechnology), anti-TEAD4 antibody (Abcam) or anti-SRF antibody (Cell Signaling) and Protein A/G agarose (GenDEPOT).

    Techniques: In Vivo, Immunohistochemistry, Infection, Expressing, shRNA, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Generated, Injection, Mouse Assay

    The transcription factors Elk-1 and SRF bind to the Mcl-1 promoter in MCF-7 cells. ( a ) Chromatin immunoprecipitation using antibodies specific to Elk-1 and SRF was performed as described in Material and methods section. Controls shown are two negative antibody controls as well as a no antibody control (protein G beads alone). Ctrl1 is an antibody against Stat-3 and Ctrl 2 is an antibody against NF-κB. Fold enrichment values were obtained by comparing the cT values for each ChIP sample to an equal amount of input DNA. Primers were designed in the last exon of the Mcl-1 gene to demonstrate specificity (RT–PCR data not shown, PCR products shown in ( b )). Data represents the mean of three independent experiments±standard error. ( b ) PCR products were run on an agarose gel following each ChIP experiment to assess reaction specificity. ( c ) Streptavidin pull-down assay to detect transcription factor binding to a 50 bp double-stranded biotin labelled probe specific to the Mcl-1 promoter region of interest. Cells were stimulated with EGF for different time periods and nuclear extracts incubated with the Mcl-1 probe. Following pull down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labelled scrambled probe was used along with competition with an unlabelled specific probe. Blots were probed with antibodies to the transcription factors Stat-3 and NF-κB to demonstrate binding specificity. ( d ) Schematic representation of the Mcl-1 gene showing approximate locations of the biotin labelled probe used for the Streptavidin pull down as well as the primers used for ChIP.

    Journal: Oncogene

    Article Title: Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer

    doi: 10.1038/onc.2010.616

    Figure Lengend Snippet: The transcription factors Elk-1 and SRF bind to the Mcl-1 promoter in MCF-7 cells. ( a ) Chromatin immunoprecipitation using antibodies specific to Elk-1 and SRF was performed as described in Material and methods section. Controls shown are two negative antibody controls as well as a no antibody control (protein G beads alone). Ctrl1 is an antibody against Stat-3 and Ctrl 2 is an antibody against NF-κB. Fold enrichment values were obtained by comparing the cT values for each ChIP sample to an equal amount of input DNA. Primers were designed in the last exon of the Mcl-1 gene to demonstrate specificity (RT–PCR data not shown, PCR products shown in ( b )). Data represents the mean of three independent experiments±standard error. ( b ) PCR products were run on an agarose gel following each ChIP experiment to assess reaction specificity. ( c ) Streptavidin pull-down assay to detect transcription factor binding to a 50 bp double-stranded biotin labelled probe specific to the Mcl-1 promoter region of interest. Cells were stimulated with EGF for different time periods and nuclear extracts incubated with the Mcl-1 probe. Following pull down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labelled scrambled probe was used along with competition with an unlabelled specific probe. Blots were probed with antibodies to the transcription factors Stat-3 and NF-κB to demonstrate binding specificity. ( d ) Schematic representation of the Mcl-1 gene showing approximate locations of the biotin labelled probe used for the Streptavidin pull down as well as the primers used for ChIP.

    Article Snippet: The following antibodies were used: rabbit anti-Mcl-1 (M8434 Sigma-Aldrich), rabbit anti-Elk-1 (ab32106 Abcam, Cambridge, MA, USA), mouse anti-SRF (MAB4369 Millipore, Billerica, MA, USA), rabbit anti-phospho-Elk-1 (Ser383) (9181, Cell Signaling, Boston, MA, USA), mouse anti-phospho-P44/42 MAPK (Thr202/Tyr204) (9106, Cell Signaling), rabbit anti-P44/42 MAPK (9102, Cell Signaling), rabbit anti-NF-κB p65 (ab7970 Abcam), mouse anti-α-tubulin (T6074 Sigma-Aldrich), rabbit-anti-Stat-3 (9132, Cell Signaling), rabbit anti-Her-2 (Dako, Mississauga, ON, Canada) and mouse anti-EGFR (Ventana, Tucson AZ, USA).

    Techniques: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Pull Down Assay, Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot

    Actin signaling modulates neuronal gene expression. A , R62D decreased, G15S increased, and S14C did not influence SRF luciferase activity in neurons. B , R62D antagonized constitutively active SRF (SRF-VP16)-induced luciferase activity. C–F , SRF-VP16 ( E ) partially rescued R62D-decreased neurite length in contrast to SRF-VP16ΔMADS ( D ) used as control (quantified in F ). G–M , R62D expression, confined to the nucleus (R62D NLS ), was recognized by its FLAG tag. R62D NLS -positive neurons ( H , J , L , arrows) showed severe structural abnormalities including decreased neurite length and lack of growth-cone filopodia. In control neurons ( K ), growth cones were collapsed by ephrin-A5 ( K , arrowheads). Contrastingly, ephrin-A5 failed to collapse a growth cone ( L , arrowhead) of an R62D NLS -expressing neuron ( L , arrow; quantified in M ). N , R62D NLS decreased SRF luciferase activity and counteracted SRF-VP16 enhanced gene activity (see B ). O , SRF-VP16 fully rescued R62D NLS -decreased neurite length. P , Cytoplasmic R62D and R62D NLS modulated expression of MAL, but not SRF. R62D NLS decreased expression of a 100 kDa MAL protein, but upregulated a MAL variant of ∼70 kDa. Cytoplasmic R62D increased abundance of the 70 kDa MAL protein, but not of 100 kDa MAL. Triplicate culture of three different mice for R62D and R62D NLS are shown. Scale bars ( C–E , G–L ), 5 μm.

    Journal: The Journal of Neuroscience

    Article Title: A Nuclear Actin Function Regulates Neuronal Motility by Serum Response Factor-Dependent Gene Transcription

    doi: 10.1523/JNEUROSCI.0333-09.2009

    Figure Lengend Snippet: Actin signaling modulates neuronal gene expression. A , R62D decreased, G15S increased, and S14C did not influence SRF luciferase activity in neurons. B , R62D antagonized constitutively active SRF (SRF-VP16)-induced luciferase activity. C–F , SRF-VP16 ( E ) partially rescued R62D-decreased neurite length in contrast to SRF-VP16ΔMADS ( D ) used as control (quantified in F ). G–M , R62D expression, confined to the nucleus (R62D NLS ), was recognized by its FLAG tag. R62D NLS -positive neurons ( H , J , L , arrows) showed severe structural abnormalities including decreased neurite length and lack of growth-cone filopodia. In control neurons ( K ), growth cones were collapsed by ephrin-A5 ( K , arrowheads). Contrastingly, ephrin-A5 failed to collapse a growth cone ( L , arrowhead) of an R62D NLS -expressing neuron ( L , arrow; quantified in M ). N , R62D NLS decreased SRF luciferase activity and counteracted SRF-VP16 enhanced gene activity (see B ). O , SRF-VP16 fully rescued R62D NLS -decreased neurite length. P , Cytoplasmic R62D and R62D NLS modulated expression of MAL, but not SRF. R62D NLS decreased expression of a 100 kDa MAL protein, but upregulated a MAL variant of ∼70 kDa. Cytoplasmic R62D increased abundance of the 70 kDa MAL protein, but not of 100 kDa MAL. Triplicate culture of three different mice for R62D and R62D NLS are shown. Scale bars ( C–E , G–L ), 5 μm.

    Article Snippet: Lysates of mock-electroporated E17 cortical neurons or electroporated with 3 μg of R62D or R62DNLS were analyzed using goat anti-MAL (1:1500; Santa Cruz), rabbit anti-MAL (a kind gift from Dr. R. Treisman), or rabbit anti-SRF antibodies (1:500; Santa Cruz).

    Techniques: Expressing, Luciferase, Activity Assay, FLAG-tag, Variant Assay, Mouse Assay

    Serum response factor (SRF) and NK3 transcription factors are required for nuclear import in smooth muscle cells (SMCs). ( a ) Immunoblots of human pulmonary artery SMCs (hSMCs) transfected with nontargeting (NT) siRNA or siRNA targeting SRF, NKX3-1 and

    Journal: Gene therapy

    Article Title: Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

    doi: 10.1038/gt.2008.83

    Figure Lengend Snippet: Serum response factor (SRF) and NK3 transcription factors are required for nuclear import in smooth muscle cells (SMCs). ( a ) Immunoblots of human pulmonary artery SMCs (hSMCs) transfected with nontargeting (NT) siRNA or siRNA targeting SRF, NKX3-1 and

    Article Snippet: Mouse monoclonal anti-SRF antibody (Millipore), rabbit polyclonal anti-Nkx3.1 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), goat polyclonal anti-Nkx3.2 antibody (Santa Cruz Biotechnologies) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnologies) were used for western blots followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL, USA).

    Techniques: Western Blot, Transfection

    Recombinant serum response factor (SRF) and Nkx3.2 are sufficient to rescue import in TC7 epithelial cells. ( a ) Immunoblots of cellular lysates reveal that SRF, NKX3-1 and NKX3-2 are expressed in human pulmonary artery smooth muscle cells (hSMCs) but

    Journal: Gene therapy

    Article Title: Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

    doi: 10.1038/gt.2008.83

    Figure Lengend Snippet: Recombinant serum response factor (SRF) and Nkx3.2 are sufficient to rescue import in TC7 epithelial cells. ( a ) Immunoblots of cellular lysates reveal that SRF, NKX3-1 and NKX3-2 are expressed in human pulmonary artery smooth muscle cells (hSMCs) but

    Article Snippet: Mouse monoclonal anti-SRF antibody (Millipore), rabbit polyclonal anti-Nkx3.1 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), goat polyclonal anti-Nkx3.2 antibody (Santa Cruz Biotechnologies) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnologies) were used for western blots followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL, USA).

    Techniques: Recombinant, Western Blot

    Engineered transcription factors targeting the FosB promoter bidirectionally regulate gene expression in NAc neurons via epigenetic manipulation. (a) Locations of FosB -ZFP binding relative to the FosB TSS. The location of functional SRF and CREB binding

    Journal: Nature neuroscience

    Article Title: Locus-Specific Epigenetic Remodeling Controls Addiction- and Depression-Related Behaviors (NN-A50213-T)

    doi: 10.1038/nn.3871

    Figure Lengend Snippet: Engineered transcription factors targeting the FosB promoter bidirectionally regulate gene expression in NAc neurons via epigenetic manipulation. (a) Locations of FosB -ZFP binding relative to the FosB TSS. The location of functional SRF and CREB binding

    Article Snippet: Sheared chromatin was incubated overnight with the following antibodies previously bound to magnetic beads (Dynabeads M-280, Life Technologies): anti-H3K9me2 antibody (AbCam ab1220 ), anti-H3K27me3 (AbCam ab6002 ), anti-H3K4me3 (EMD Millipore 17-614 ), H3K9/14ac (EMD Millipore 17-615 ), anti-H3K9me3 (EMD Millipore 07-442 ), anti-HP1α (EMD Millipore 05-689 ), anti-CREB (EMD Millipore 17-600 ), anti-phospho-CREB (EMD Millipore 17-10131 ), anti-phospho-SRF (AbCam ab53130).

    Techniques: Expressing, Binding Assay, Functional Assay

    Increased responsiveness to SRF in the intron 14 from SHR. (A) Analysis of intron 14 in other normotensive and hypertensive rat strains. Stroke-prone SHR (SHRSP) also contains the 12-base pair insertion, whereas normotensive SD and WKY rats do not. (B) Activity of intron 14 from WKY (WKY-pMK) rats, SD (SD-pMK) rats, and SHR (SHR-pMK) was analyzed using luciferase reporter gene assays. Although coexpression with 50 ng of the SRF plasmid increased the activities of all three promoters, it increased SHR promoter activity much more than the activities of the WKY and SD promoters (7.2-, 2.7-, and 2.1-fold compared with control cells transfected with 50 ng of pcDNA 3.1, respectively). The means ± SE are shown. *p

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-04-0353

    Figure Lengend Snippet: Increased responsiveness to SRF in the intron 14 from SHR. (A) Analysis of intron 14 in other normotensive and hypertensive rat strains. Stroke-prone SHR (SHRSP) also contains the 12-base pair insertion, whereas normotensive SD and WKY rats do not. (B) Activity of intron 14 from WKY (WKY-pMK) rats, SD (SD-pMK) rats, and SHR (SHR-pMK) was analyzed using luciferase reporter gene assays. Although coexpression with 50 ng of the SRF plasmid increased the activities of all three promoters, it increased SHR promoter activity much more than the activities of the WKY and SD promoters (7.2-, 2.7-, and 2.1-fold compared with control cells transfected with 50 ng of pcDNA 3.1, respectively). The means ± SE are shown. *p

    Article Snippet: A band shift was detected when nuclear extracts were incubated with WKY or SHR oligonucleotides, and a supershifted band was detected when anti-SRF antibodies were added to the reaction ( A).

    Techniques: Activity Assay, Luciferase, Plasmid Preparation, Transfection

    Analysis of introns 13 and 14. (A and B) Homology analysis of the introns 13 (A) or 14 (B) among species. Darkness gradients from black to white represent homology from 100 to 0%. Note that the TATA box and the CArG box are conserved in all the species. (C) TBP binds to intron 14 of the MYLK gene. Purified TBP was incubated with 32 P-labeled intron 14 from WKY and analyzed using an EMSA. TBP induced a concentration-dependent appearance of a slower migrating band (arrow). A 10-fold excess of cold, nonradioactive competitor eliminated this band. (D) ChIP assays were performed with antibodies to SRF or RNA polymerase II. Nonspecific IgG was used as a negative control (IgG). Input chromatin (0.2, 0.02, and 0.002%) and immunoprecipitated DNA (4, 0.4, and 0.04%) were amplified with primers specific for the |gb-globin promoter, SM-MHC promoter or intron 14 of MYLK gene. SRF and RNA polymerase II are not present in the β-globin promoter. However, SRF bound to the SM-MHC promoter and to intron 14 of the MYCK gene in VSMC from WKY rats. RNA polymerase II also bound to the intron 14. (E) Serial deletion of intron 14 isolated from WKY rats. Deletion fragments were isolated using PCR and cloned into the pGL 3 -Basic luciferase reporter vector. The numbers indicate the location of the first nucleotide in each fragment, whereas the black bars indicate relative luciferase activities. CT, CArG, and TATA represent CT repeats, CArG box, and TATA box, respectively. The means ± SE from four experiments are shown.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-04-0353

    Figure Lengend Snippet: Analysis of introns 13 and 14. (A and B) Homology analysis of the introns 13 (A) or 14 (B) among species. Darkness gradients from black to white represent homology from 100 to 0%. Note that the TATA box and the CArG box are conserved in all the species. (C) TBP binds to intron 14 of the MYLK gene. Purified TBP was incubated with 32 P-labeled intron 14 from WKY and analyzed using an EMSA. TBP induced a concentration-dependent appearance of a slower migrating band (arrow). A 10-fold excess of cold, nonradioactive competitor eliminated this band. (D) ChIP assays were performed with antibodies to SRF or RNA polymerase II. Nonspecific IgG was used as a negative control (IgG). Input chromatin (0.2, 0.02, and 0.002%) and immunoprecipitated DNA (4, 0.4, and 0.04%) were amplified with primers specific for the |gb-globin promoter, SM-MHC promoter or intron 14 of MYLK gene. SRF and RNA polymerase II are not present in the β-globin promoter. However, SRF bound to the SM-MHC promoter and to intron 14 of the MYCK gene in VSMC from WKY rats. RNA polymerase II also bound to the intron 14. (E) Serial deletion of intron 14 isolated from WKY rats. Deletion fragments were isolated using PCR and cloned into the pGL 3 -Basic luciferase reporter vector. The numbers indicate the location of the first nucleotide in each fragment, whereas the black bars indicate relative luciferase activities. CT, CArG, and TATA represent CT repeats, CArG box, and TATA box, respectively. The means ± SE from four experiments are shown.

    Article Snippet: A band shift was detected when nuclear extracts were incubated with WKY or SHR oligonucleotides, and a supershifted band was detected when anti-SRF antibodies were added to the reaction ( A).

    Techniques: Purification, Incubation, Labeling, Concentration Assay, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Isolation, Polymerase Chain Reaction, Clone Assay, Luciferase, Plasmid Preparation

    Isolation and analysis of intron 14 of the MYLK gene. (A) Exon–intron structure of the rat smMLCK gene. The translation start site of the smMLCK in exon 15 is shown (ATG). (B) DNA sequences of intron 14 from SHR and WKY rats. Intron 14 was isolated from genomic DNA from SHR and WKY rats using PCR. DNA sequences of the 5′ and 3′ primers are shown (italics, underlined). The transcription start site (+1) of smMLCK was identified using primer extension and 5′ RACE (Supplemental Figure 1). Analysis of transcription elements using the Transcription Element Search System identified > 55 elements, including SRF (CArG box) and TBP (TATA box) binding sites. Comparison of these sequences revealed the presence of a 12-base pair insertion (Insertion) in the SHR sequence not found in the WKY sequence. WKY also contains four different single nucleotide polymorphisms (bold, caps) compared with SHR and other rats.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-04-0353

    Figure Lengend Snippet: Isolation and analysis of intron 14 of the MYLK gene. (A) Exon–intron structure of the rat smMLCK gene. The translation start site of the smMLCK in exon 15 is shown (ATG). (B) DNA sequences of intron 14 from SHR and WKY rats. Intron 14 was isolated from genomic DNA from SHR and WKY rats using PCR. DNA sequences of the 5′ and 3′ primers are shown (italics, underlined). The transcription start site (+1) of smMLCK was identified using primer extension and 5′ RACE (Supplemental Figure 1). Analysis of transcription elements using the Transcription Element Search System identified > 55 elements, including SRF (CArG box) and TBP (TATA box) binding sites. Comparison of these sequences revealed the presence of a 12-base pair insertion (Insertion) in the SHR sequence not found in the WKY sequence. WKY also contains four different single nucleotide polymorphisms (bold, caps) compared with SHR and other rats.

    Article Snippet: A band shift was detected when nuclear extracts were incubated with WKY or SHR oligonucleotides, and a supershifted band was detected when anti-SRF antibodies were added to the reaction ( A).

    Techniques: Isolation, Polymerase Chain Reaction, Binding Assay, Sequencing

    Increased SRF binding to the CArG box in the SHR promoter. (A) Two oligonucleotides representing regions of the smMLCK promoters containing the CT repeats and CArG box were synthesized. The only difference between them was the presence of a 12-base pair insertion in the SHR oligonucleotides. A WKY oligonucleotide without the CArG box was also synthesized and used as a negative control (ΔCArG). Nuclear extracts were isolated from cells expressing SRF (NE). For competition titration assays, three different amounts of the cold competitors (5-, 15-, or 50-fold to 32 P-labeled probe) were incubated with the nuclear extracts and 32 P-labeled WKY or SHR oligonucleotides. Autoradiography demonstrated the presence of slower migrating bands (arrow) that decreased in intensity as the concentration of the competitors was increased. These bands were always more intense when incubated with the oligonucleotides representing the SHR promoter, and SRF antibodies supershifted this band (arrowhead). (B) In vitro ChIP assays. Cross-linked chromatin from VSMCs of SHR or WKY rats was immunoprecipitated with antibodies to SRF. Input chromatin (0.1, 0.01, and 0.001%) and immunoprecipitated DNA (2, 0.2, and 0.02%) were amplified with primers specific for intron 14. (C) In vivo ChIP assays. Cross-linked chromatin was isolated from aortas removed from SHR or WKY rats. Input chromatin (1, 0.1, and 0.01%) and immunoprecipitated DNA (20, 2, and 0.2%) were amplified with the same primers used for the in vitro ChIP assays. Comparing the relative abundance of the signals confirmed increased binding of SRF to the intron in SHR compared with the intron in WKY rats.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-04-0353

    Figure Lengend Snippet: Increased SRF binding to the CArG box in the SHR promoter. (A) Two oligonucleotides representing regions of the smMLCK promoters containing the CT repeats and CArG box were synthesized. The only difference between them was the presence of a 12-base pair insertion in the SHR oligonucleotides. A WKY oligonucleotide without the CArG box was also synthesized and used as a negative control (ΔCArG). Nuclear extracts were isolated from cells expressing SRF (NE). For competition titration assays, three different amounts of the cold competitors (5-, 15-, or 50-fold to 32 P-labeled probe) were incubated with the nuclear extracts and 32 P-labeled WKY or SHR oligonucleotides. Autoradiography demonstrated the presence of slower migrating bands (arrow) that decreased in intensity as the concentration of the competitors was increased. These bands were always more intense when incubated with the oligonucleotides representing the SHR promoter, and SRF antibodies supershifted this band (arrowhead). (B) In vitro ChIP assays. Cross-linked chromatin from VSMCs of SHR or WKY rats was immunoprecipitated with antibodies to SRF. Input chromatin (0.1, 0.01, and 0.001%) and immunoprecipitated DNA (2, 0.2, and 0.02%) were amplified with primers specific for intron 14. (C) In vivo ChIP assays. Cross-linked chromatin was isolated from aortas removed from SHR or WKY rats. Input chromatin (1, 0.1, and 0.01%) and immunoprecipitated DNA (20, 2, and 0.2%) were amplified with the same primers used for the in vitro ChIP assays. Comparing the relative abundance of the signals confirmed increased binding of SRF to the intron in SHR compared with the intron in WKY rats.

    Article Snippet: A band shift was detected when nuclear extracts were incubated with WKY or SHR oligonucleotides, and a supershifted band was detected when anti-SRF antibodies were added to the reaction ( A).

    Techniques: Binding Assay, Synthesized, Negative Control, Isolation, Expressing, Titration, Labeling, Incubation, Autoradiography, Concentration Assay, In Vitro, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, In Vivo

    Co-expression of αSMA and SRF in part of stromal cells originated from bone marrow-derived cells. ( A ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed αSMA (indicated by arrows) in a carcinogen-induced small intestinal tumor. ( B ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed SRF (indicated by arrows) in the small intestinal tumor. A SRF(+) cell without GFP expression is indicated by a filled arrow.

    Journal: PLoS ONE

    Article Title: Bone Marrow-Derived Cells May Not Be the Original Cells for Carcinogen-Induced Mouse Gastrointestinal Carcinomas

    doi: 10.1371/journal.pone.0079615

    Figure Lengend Snippet: Co-expression of αSMA and SRF in part of stromal cells originated from bone marrow-derived cells. ( A ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed αSMA (indicated by arrows) in a carcinogen-induced small intestinal tumor. ( B ) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed SRF (indicated by arrows) in the small intestinal tumor. A SRF(+) cell without GFP expression is indicated by a filled arrow.

    Article Snippet: For dual fluorescence immuno-staining, slides were incubated with primary antibody against CD45 (1∶100; B0848, Anbobio, San Francisco, CA), α-smooth muscle actin (αSMA) (1∶250; AB7817, Abcam) or serum response factor (SRF) (1∶75; AB53147, Abcam) at 4°C overnight, then with TRITC-conjugated secondary antibody (Abcam) for 1 hour at room temperature and observed as described above.

    Techniques: Expressing, Derivative Assay

    Future subplate genes are expressed in RELN-negative preplate and subplate neurons. ( a ) Genes expressed in future subplate neurons categorized by function. ( b – h ) Double immunofluorescent staining for RELN (red) and marker proteins for future subplate candidate genes (green) in coronal cryostat sections of wild-type E12.5 ( b , c ) and E14.5 ( c – h ) brains. Hpca ( c , e ) and EAAC1 ( b , d ) are expressed in RELN-negative preplate cells and in subplate cell bodies. At E14.5, SRF ( f ) and RyR1 ( h ) are expressed in RELN-positive marginal zone neurons, as well as in intermediate zone fibers originating in the subplate, whereas Sez6 -driven EGFP ( g ) is expressed in subplate and cortical plate neurons, but not in RELN-positive cells. Scale bars = 50 μm.

    Journal: Cerebral Cortex (New York, NY)

    Article Title: Gene Expression Profiling of Preplate Neurons Destined for the Subplate: Genes Involved in Transcription, Axon Extension, Neurotransmitter Regulation, Steroid Hormone Signaling, and Neuronal Survival

    doi: 10.1093/cercor/bhp034

    Figure Lengend Snippet: Future subplate genes are expressed in RELN-negative preplate and subplate neurons. ( a ) Genes expressed in future subplate neurons categorized by function. ( b – h ) Double immunofluorescent staining for RELN (red) and marker proteins for future subplate candidate genes (green) in coronal cryostat sections of wild-type E12.5 ( b , c ) and E14.5 ( c – h ) brains. Hpca ( c , e ) and EAAC1 ( b , d ) are expressed in RELN-negative preplate cells and in subplate cell bodies. At E14.5, SRF ( f ) and RyR1 ( h ) are expressed in RELN-positive marginal zone neurons, as well as in intermediate zone fibers originating in the subplate, whereas Sez6 -driven EGFP ( g ) is expressed in subplate and cortical plate neurons, but not in RELN-positive cells. Scale bars = 50 μm.

    Article Snippet: Primary antibodies used were: mouse anti-Cajal–Retzius cell marker reelin (RELN) (1:1000, MAB5364, Chemicon, Billerica, MA), rabbit anti-GFP (1:2000, Molecular Probes, Carlsbad, CA), rabbit anti-calretinin (1:2000, Swant, Bellinzona, Switzerland), rabbit anti-hippocalcin (1:2000, Abcam, Cambridge, MA), goat anti-EAAC1 (1:2000, Chemicon), rabbit anti-Ryr1 (1:1000, Chemicon), rabbit anti-serum response factor (SRF) (1:100, Abcam), rat anti-L1 (1:20, a gift from Dr J. Trotter), mouse IgM anti-TAG1 (1:2, gift from Dr Jane Dodd, Columbia University, NY).

    Techniques: Staining, Marker

    mDia2-SRF signaling is involved in the regulation of HSPC engraftment. a Immunofluorescence staining of HSPCs with F-actin (red) and α-tubulin. White arrows: F-actin protrusions. Yellow arrows: polarization axis to determine the staining intensity in b . The experiments were repeated three times with similar results. b F-actin and α-tubulin staining intensity across the yellow arrows from a . c Quantitative analyses of F-actin staining intensity from a . N = 17 cells in mDia2 fl/fl group. N = 13 cells in mDia2 fl/fl Vav-Cre group. d Quantitative analyses of the percentage of cells with F-actin protrusions in a . N = 5 fields in mDia2 fl/fl group. N = 6 fields in mDia2 fl/fl Vav-Cre group. e Quantitative analysis of the percentages of MAL nuclear localization in c-Kit+ HSPCs incubated with or without FBS for 15 min. mDia2 fl/fl no FBS: N = 71 cells from 17 random fields; mDia2 fl/fl Vav-Cre no FBS: N = 80 cells from 15 random fields; mDia2 fl/fl with 10% FBS: N = 56 cells from 12 random fields; mDia2 fl/fl Vav-Cre with 10% FBS: N = 108 cells from 19 random fields. Data are from three independent experiments. f Quantitative RT-PCR of the indicated SRF target genes in c-Kit+ HSPCs from the indicated mice. g Quantitative RT-PCR of the indicated integrins in c-Kit+ HSPCs from the indicated mice. The experiments were performed in triplicate from n = 5 mice per group in f , g . h Quantitative analyses of the surface expression level of beta2 integrins using flow cytometry in the indicated HSPC populations. n = 2 mice per group. i Representative flow cytometry plots showing the expression of beta2 integrins in ex vivo-cultured c-Kit+ HSPCs. j Quantitative analyses of i . n = 2 mice per group. k In vitro adhesion assay of the cultured c-kit+ HSPCs from the indicated mice on extracellular matrix protein coated coverslips. Data are presented as adhesion ability relative to the control cells. mDia2 fl/fl , Collagen: 25 fields, Fibronectin: 20 fields, poly- d -lysine: 15 fields. mDia2 fl/fl Vav-Cre, Collagen: 10 fields, Fibronectin: 14 fields, poly- d -lysine: 9 fields. l In vitro adhesion assay of indicated c-kit+ HSPCs cultured on ICAM-1-coated coverslips. Error bars represent the SEM of the mean. * p

    Journal: Nature Communications

    Article Title: Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment

    doi: 10.1038/s41467-020-16911-4

    Figure Lengend Snippet: mDia2-SRF signaling is involved in the regulation of HSPC engraftment. a Immunofluorescence staining of HSPCs with F-actin (red) and α-tubulin. White arrows: F-actin protrusions. Yellow arrows: polarization axis to determine the staining intensity in b . The experiments were repeated three times with similar results. b F-actin and α-tubulin staining intensity across the yellow arrows from a . c Quantitative analyses of F-actin staining intensity from a . N = 17 cells in mDia2 fl/fl group. N = 13 cells in mDia2 fl/fl Vav-Cre group. d Quantitative analyses of the percentage of cells with F-actin protrusions in a . N = 5 fields in mDia2 fl/fl group. N = 6 fields in mDia2 fl/fl Vav-Cre group. e Quantitative analysis of the percentages of MAL nuclear localization in c-Kit+ HSPCs incubated with or without FBS for 15 min. mDia2 fl/fl no FBS: N = 71 cells from 17 random fields; mDia2 fl/fl Vav-Cre no FBS: N = 80 cells from 15 random fields; mDia2 fl/fl with 10% FBS: N = 56 cells from 12 random fields; mDia2 fl/fl Vav-Cre with 10% FBS: N = 108 cells from 19 random fields. Data are from three independent experiments. f Quantitative RT-PCR of the indicated SRF target genes in c-Kit+ HSPCs from the indicated mice. g Quantitative RT-PCR of the indicated integrins in c-Kit+ HSPCs from the indicated mice. The experiments were performed in triplicate from n = 5 mice per group in f , g . h Quantitative analyses of the surface expression level of beta2 integrins using flow cytometry in the indicated HSPC populations. n = 2 mice per group. i Representative flow cytometry plots showing the expression of beta2 integrins in ex vivo-cultured c-Kit+ HSPCs. j Quantitative analyses of i . n = 2 mice per group. k In vitro adhesion assay of the cultured c-kit+ HSPCs from the indicated mice on extracellular matrix protein coated coverslips. Data are presented as adhesion ability relative to the control cells. mDia2 fl/fl , Collagen: 25 fields, Fibronectin: 20 fields, poly- d -lysine: 15 fields. mDia2 fl/fl Vav-Cre, Collagen: 10 fields, Fibronectin: 14 fields, poly- d -lysine: 9 fields. l In vitro adhesion assay of indicated c-kit+ HSPCs cultured on ICAM-1-coated coverslips. Error bars represent the SEM of the mean. * p

    Article Snippet: Briefly, chromatin was incubated with anti-SRF antibody (2C5, Catalog #61386, Active Motif), anti-MAL antibody (sc-390324, Santa Cruz), or anti-GFP (B2, Catalog #sc-9996, Santa Cruz) at 4 °C overnight.

    Techniques: Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Mouse Assay, Expressing, Flow Cytometry, Ex Vivo, Cell Culture, In Vitro, Cell Adhesion Assay

    (A) Effect of PIAS1 on the formation of CArG-SRF complex containing myocardin by EMSA. Rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and nuclear extracts were prepared and incubated with a 32 P labeled 95-bp probe corresponding to the −137 to −42 bp region of the SM α-actin gene. Nuclear extracts from bovine aortic EC were used as a control. Arrowheads A and B indicate the DNA protein complex from EC and SMC, respectively. Polyclonal antibody against SRF or control IgG was added for supershift. (B) ChIP assays. Cultured rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and SRF binding to the CArG region within intact chromatin was examined by ChIP assays as described in Materials and Methods. Quantitative PCR was used to detect CArG-containing regions of the SM α-actin promoter in chromatin fragments immunoprecipitated with anti-SRF antibody. An n -fold enrichment of 100 was assigned to SRF association in cells overexpressing control (αGFP). Values present the means ± standard errors of the mean. *, P of

    Journal: Molecular and Cellular Biology

    Article Title: PIAS1 Activates the Expression of Smooth Muscle Cell Differentiation Marker Genes by Interacting with Serum Response Factor and Class I Basic Helix-Loop-Helix Proteins

    doi: 10.1128/MCB.25.18.8009-8023.2005

    Figure Lengend Snippet: (A) Effect of PIAS1 on the formation of CArG-SRF complex containing myocardin by EMSA. Rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and nuclear extracts were prepared and incubated with a 32 P labeled 95-bp probe corresponding to the −137 to −42 bp region of the SM α-actin gene. Nuclear extracts from bovine aortic EC were used as a control. Arrowheads A and B indicate the DNA protein complex from EC and SMC, respectively. Polyclonal antibody against SRF or control IgG was added for supershift. (B) ChIP assays. Cultured rat aortic SMC were transfected with siRNA oligonucleotides specific for PIAS1 (αPIAS1) or GFP (αGFP), and SRF binding to the CArG region within intact chromatin was examined by ChIP assays as described in Materials and Methods. Quantitative PCR was used to detect CArG-containing regions of the SM α-actin promoter in chromatin fragments immunoprecipitated with anti-SRF antibody. An n -fold enrichment of 100 was assigned to SRF association in cells overexpressing control (αGFP). Values present the means ± standard errors of the mean. *, P of

    Article Snippet: Western blot analysis was performed according to standard procedures using the following primary antibodies: monoclonal anti-Flag antibody (M2; Sigma), polyclonal anti-E12 antibody (Santa Cruz), and polyclonal anti-SRF antibody (Santa Cruz).

    Techniques: Transfection, Incubation, Labeling, Chromatin Immunoprecipitation, Cell Culture, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation