anti rarα Santa Cruz Biotechnology Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 89
    Santa Cruz Biotechnology anti rarα
    <t>PLZF-RARα</t> promotes cell proliferation but does not induce apoptosis. A, FACS analysis of HEK293 and HCT116 cells transfected with either a pcDNA3 or pSG5-PLZF-RARα plasmid. B , MTT assay of cell proliferation. HEK293 and HCT116 cells transfected
    Anti Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rarα/product/Santa Cruz Biotechnology
    Average 89 stars, based on 231 article reviews
    Price from $9.99 to $1999.99
    anti rarα - by Bioz Stars, 2020-05
    89/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology rabbit anti rarα
    A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing <t>RARα</t> or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.
    Rabbit Anti Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rarα/product/Santa Cruz Biotechnology
    Average 88 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    rabbit anti rarα - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology goat anti rarα
    A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing <t>RARα</t> or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.
    Goat Anti Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rarα/product/Santa Cruz Biotechnology
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    goat anti rarα - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology human rarα
    In vitro differentiation of myeloid progenitors from mCG <t>PML-RARα</t> cells with or without neutrophil elastase. (Left) Mean cell numbers and standard deviations over time in culture. Data represent cultures of bone marrow from one of two independent
    Human Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human rarα/product/Santa Cruz Biotechnology
    Average 88 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    human rarα - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology c 20 anti rarα antibody
    In vitro differentiation of myeloid progenitors from mCG <t>PML-RARα</t> cells with or without neutrophil elastase. (Left) Mean cell numbers and standard deviations over time in culture. Data represent cultures of bone marrow from one of two independent
    C 20 Anti Rarα Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c 20 anti rarα antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    c 20 anti rarα antibody - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology polyclonal anti rarα peptide antibodies
    In vitro differentiation of myeloid progenitors from mCG <t>PML-RARα</t> cells with or without neutrophil elastase. (Left) Mean cell numbers and standard deviations over time in culture. Data represent cultures of bone marrow from one of two independent
    Polyclonal Anti Rarα Peptide Antibodies, supplied by Santa Cruz Biotechnology, 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/polyclonal anti rarα peptide antibodies/product/Santa Cruz Biotechnology
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti rarα peptide antibodies - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology rabbit polyclonal anti rarα antibody
    <t>RARα</t> interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit <t>polyclonal</t> anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.
    Rabbit Polyclonal Anti Rarα Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti rarα antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti rarα antibody - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology polyclonal anti rarα
    <t>RARα</t> interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit <t>polyclonal</t> anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.
    Polyclonal Anti Rarα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti rarα/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti rarα - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology rarα c 20
    <t>RARα</t> interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit <t>polyclonal</t> anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.
    Rarα C 20, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rarα c 20/product/Santa Cruz Biotechnology
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    rarα c 20 - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology rabbit anti rarα primary antibody
    <t>RARα</t> interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit <t>polyclonal</t> anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.
    Rabbit Anti Rarα Primary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rarα primary antibody/product/Santa Cruz Biotechnology
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    rabbit anti rarα primary antibody - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology rarα primary ab
    Model for <t>RARα</t> dependent epigenetic regulation. ( A ) Ligand-independent binding of RARα/RXRα heterodimers to the Mest promoter region is required to maintain transcriptionally permissive histone modifications (H3K4me3 and H3K9/K14ac) and relatively low methylation levels of promoter CpG islands (upper panel). Knockout of RARα results in higher levels of Mest promoter methylation, loss of permissive histone modifications, and gain of repressive histone modifications (lower panel). ( B ) Actively transcribed genes display relative low levels of promoter methylation and high levels of H3K9/K14ac and H3K4me3 (Mest and Tex13 in Wt, Stmn2 and Slc38a4 in RARα −/− ), whereas silenced genes display high levels of promoter methylation and, if paternally expressed, high H3K9me3 levels (Slc38a4 in Wt, Mest in RARα −/− ). Transcriptional start sites (TSS) and relative transcriptional activities are indicated by the arrows and arrow sizes, respectively. The relative levels of CpG promoter methylation are denoted by black lollipops (the CpG-rich regions assessed each span the TSS but for clarity are drawn upstream of the TSS).
    Rarα Primary Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rarα primary ab/product/Santa Cruz Biotechnology
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rarα primary ab - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology rabbit anti human rarα antibody
    miR-126 is not regulated by flow-eliciting <t>RARα/RXRα</t> or HDAC-3/5/7 signaling in ECs. ( A ) ECs were kept under static condition or exposed to flows over the 24-h test period, and their miR-126 expression was determined by qPCR. ( B and C
    Rabbit Anti Human Rarα Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti human rarα antibody/product/Santa Cruz Biotechnology
    Average 91 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    rabbit anti human rarα antibody - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    80
    Santa Cruz Biotechnology anti rarα polyclonal antibody c 20
    pRB colocalizes with PML within nuclear bodies and is delocalized by <t>PML-RARα</t> expression. (A) Immunofluorescence experiments using an anti-pRB monoclonal antibody (α-RB; aRB1C1) revealed with a rhodamine-conjugated anti-mouse antibody and an anti-PML <t>polyclonal</t> antibody (α-PML; 2912A) revealed with an FITC-conjugated anti-rabbit antibody were performed with IB4 and U937 cells. Superimposition of PML and pRB staining is shown in the righthand panels, indicating colocalization of PML and pRB within NBs in both cell lines. (B) Similar experiments were performed with the U937 PR9 clone before and after induction of PML-RARα expression with ZnSO 4 . Superimposition of PML and pRB staining is shown in the righthand panels, revealing that pRB is delocalized into the PML-RARα microspeckles by the expression of the fusion protein. The anti-RB antibody aRB1C1 does not cross-react with PML-RARα, as revealed by Western blotting experiments and immunoprecipitations of in vitro-translated PML-RARα polypeptides (our unpublished results).
    Anti Rarα Polyclonal Antibody C 20, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rarα polyclonal antibody c 20/product/Santa Cruz Biotechnology
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti rarα polyclonal antibody c 20 - by Bioz Stars, 2020-05
    80/100 stars
      Buy from Supplier

    85
    Santa Cruz Biotechnology rarα specific antibody sc551
    pRB colocalizes with PML within nuclear bodies and is delocalized by <t>PML-RARα</t> expression. (A) Immunofluorescence experiments using an anti-pRB monoclonal antibody (α-RB; aRB1C1) revealed with a rhodamine-conjugated anti-mouse antibody and an anti-PML <t>polyclonal</t> antibody (α-PML; 2912A) revealed with an FITC-conjugated anti-rabbit antibody were performed with IB4 and U937 cells. Superimposition of PML and pRB staining is shown in the righthand panels, indicating colocalization of PML and pRB within NBs in both cell lines. (B) Similar experiments were performed with the U937 PR9 clone before and after induction of PML-RARα expression with ZnSO 4 . Superimposition of PML and pRB staining is shown in the righthand panels, revealing that pRB is delocalized into the PML-RARα microspeckles by the expression of the fusion protein. The anti-RB antibody aRB1C1 does not cross-react with PML-RARα, as revealed by Western blotting experiments and immunoprecipitations of in vitro-translated PML-RARα polypeptides (our unpublished results).
    Rarα Specific Antibody Sc551, supplied by Santa Cruz Biotechnology, 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/rarα specific antibody sc551/product/Santa Cruz Biotechnology
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    rarα specific antibody sc551 - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    Image Search Results


    PLZF-RARα promotes cell proliferation but does not induce apoptosis. A, FACS analysis of HEK293 and HCT116 cells transfected with either a pcDNA3 or pSG5-PLZF-RARα plasmid. B , MTT assay of cell proliferation. HEK293 and HCT116 cells transfected

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα promotes cell proliferation but does not induce apoptosis. A, FACS analysis of HEK293 and HCT116 cells transfected with either a pcDNA3 or pSG5-PLZF-RARα plasmid. B , MTT assay of cell proliferation. HEK293 and HCT116 cells transfected

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: FACS, Transfection, Plasmid Preparation, MTT Assay

    Transcription repression of CDKN1A by PLZF-RARα can be independent of p53. A , transcription assays. HCT116 p53 +/+ and p53 −/− cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: Transcription repression of CDKN1A by PLZF-RARα can be independent of p53. A , transcription assays. HCT116 p53 +/+ and p53 −/− cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Hypothetical model for transcriptional regulation of CDKN1A and TP53 by PLZF-RARα and post-translational ubiquitination of p53. A , PLZF-RARα represses all four genes in the p53 pathway. ⊢, transcriptional repression; ↔,

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: Hypothetical model for transcriptional regulation of CDKN1A and TP53 by PLZF-RARα and post-translational ubiquitination of p53. A , PLZF-RARα represses all four genes in the p53 pathway. ⊢, transcriptional repression; ↔,

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques:

    PLZF-RARα stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A , MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RARα

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A , MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RARα

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: DNA Methylation Assay, MTT Assay, Transfection

    PLZF-RARα represses CDKN1A gene transcription through binding competition with p53, TP53 transcriptional repression, and increased p53 ubiquitination. A , structure of the human CDKN1A gene promoter. The arrows at the p53 binding elements indicate

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses CDKN1A gene transcription through binding competition with p53, TP53 transcriptional repression, and increased p53 ubiquitination. A , structure of the human CDKN1A gene promoter. The arrows at the p53 binding elements indicate

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Binding Assay

    PLZF-RARα represses the transcription of p53 pathway genes in HEK293 cells. A, transient transcription assays for the ARF, MDM2, TP53 , and CDKN1A genes of the p53 pathway. The PLZF-RARα expression vector and promoter-luciferase fusion

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses the transcription of p53 pathway genes in HEK293 cells. A, transient transcription assays for the ARF, MDM2, TP53 , and CDKN1A genes of the p53 pathway. The PLZF-RARα expression vector and promoter-luciferase fusion

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Expressing, Plasmid Preparation, Luciferase

    PLZF-RARα represses the transcription of CDKN1A by binding to its RARE in vitro and in vivo . A , transcription assay for the CDKN1A promoter in the presence of PLZF-RARα and ATRA or EtOH control in HEK293 cells. Cells were transiently co-transfected

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses the transcription of CDKN1A by binding to its RARE in vitro and in vivo . A , transcription assay for the CDKN1A promoter in the presence of PLZF-RARα and ATRA or EtOH control in HEK293 cells. Cells were transiently co-transfected

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Binding Assay, In Vitro, In Vivo, Transfection

    Proliferation of HL-60 cells is increased by ectopic PLZF-RARα and decreased by TSA, 5-aza-2′-deoxycytidine, ATRA, or any combination thereof. Transcriptional repression of CDKN1A by PLZF-RARα is derepressed by the reagents. A–D

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: Proliferation of HL-60 cells is increased by ectopic PLZF-RARα and decreased by TSA, 5-aza-2′-deoxycytidine, ATRA, or any combination thereof. Transcriptional repression of CDKN1A by PLZF-RARα is derepressed by the reagents. A–D

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques:

    PLZF-RARα represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A , structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B , qChIP assays showing

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A , structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B , qChIP assays showing

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: DNA Methylation Assay, Polymerase Chain Reaction, Binding Assay

    PLZF-RARα represses CDKN1A transcription by competing with Sp1 for binding to proximal promoter GC-boxes 3, 4, and 5/6, in vitro and in vivo . A , structure of the human CDKN1A promoter. The arrows indicate the binding positions of the qChIP oligonucleotide

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses CDKN1A transcription by competing with Sp1 for binding to proximal promoter GC-boxes 3, 4, and 5/6, in vitro and in vivo . A , structure of the human CDKN1A promoter. The arrows indicate the binding positions of the qChIP oligonucleotide

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Binding Assay, In Vitro, In Vivo

    PLZF-RARα represses transcriptional activation of CDKN1A by p53. A , transient transcription assays. HCT116 cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc (−2.3 kb) reporter plasmid,

    Journal: The Journal of Biological Chemistry

    Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes *

    doi: 10.1074/jbc.M113.538777

    Figure Lengend Snippet: PLZF-RARα represses transcriptional activation of CDKN1A by p53. A , transient transcription assays. HCT116 cells were transiently co-transfected with a PLZF-RARα expression vector and a pGL2-CDKN1A-Luc (−2.3 kb) reporter plasmid,

    Article Snippet: The degree of PLZF-RARα binding to p53RE-1, -2 and RARE, and Sp1-binding sites was analyzed by ChIP assays using an anti-PLZF, anti-RARα, and anti-HA tag antibody (Santa Cruz).

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation

    RARα is involved in GFAP expression. A–C , HCN-B27 adult rat hippocampal precursor cells were treated for 2 days with different combinations of gliogenic factors (all- trans -retinoic acid ( R ), 1 μ m ; bone morphogenetic protein-2 (

    Journal: The Journal of Biological Chemistry

    Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression

    doi: 10.1074/jbc.M110.170274

    Figure Lengend Snippet: RARα is involved in GFAP expression. A–C , HCN-B27 adult rat hippocampal precursor cells were treated for 2 days with different combinations of gliogenic factors (all- trans -retinoic acid ( R ), 1 μ m ; bone morphogenetic protein-2 (

    Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

    Techniques: Expressing

    PI3K controls the main events leading to astrogliogenesis. A , specific PI3K inhibitor LY294002 ( LY , 10 μ m ) completely blocks both cytokine-induced astrogliogenesis and the synergism between retinoic acid and cytokines. The specific RARα

    Journal: The Journal of Biological Chemistry

    Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression

    doi: 10.1074/jbc.M110.170274

    Figure Lengend Snippet: PI3K controls the main events leading to astrogliogenesis. A , specific PI3K inhibitor LY294002 ( LY , 10 μ m ) completely blocks both cytokine-induced astrogliogenesis and the synergism between retinoic acid and cytokines. The specific RARα

    Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

    Techniques:

    PI3K controls the association between RARα, p300/CBP, Stat3, and Smad. A , knocking down the expression of p300/CBP by siRNA partially prevents the induction of GFAP expression by retinoic acid and/or cytokines and decreases the expression of RARα.

    Journal: The Journal of Biological Chemistry

    Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression

    doi: 10.1074/jbc.M110.170274

    Figure Lengend Snippet: PI3K controls the association between RARα, p300/CBP, Stat3, and Smad. A , knocking down the expression of p300/CBP by siRNA partially prevents the induction of GFAP expression by retinoic acid and/or cytokines and decreases the expression of RARα.

    Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

    Techniques: Expressing

    RARα is also involved in cytokine-induced GFAP expression. A , constructs encoding luciferase under the control of serial deletions of the rat GFAP promoter and of the full-length promoter with the putative RARE (AGGTCA) deleted. This putative

    Journal: The Journal of Biological Chemistry

    Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression

    doi: 10.1074/jbc.M110.170274

    Figure Lengend Snippet: RARα is also involved in cytokine-induced GFAP expression. A , constructs encoding luciferase under the control of serial deletions of the rat GFAP promoter and of the full-length promoter with the putative RARE (AGGTCA) deleted. This putative

    Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

    Techniques: Expressing, Construct, Luciferase

    Cytokines potentiate the retinoic acid GFAP pathway. A–D , BMP-2 and LIF enhance the expression of RARα, which is necessary for retinoic acid-induced GFAP synthesis. On the other hand, retinoic acid ( R ) has no effect on the key phosphorylation

    Journal: The Journal of Biological Chemistry

    Article Title: Synergistic Effect of Retinoic Acid and Cytokines on the Regulation of Glial Fibrillary Acidic Protein Expression

    doi: 10.1074/jbc.M110.170274

    Figure Lengend Snippet: Cytokines potentiate the retinoic acid GFAP pathway. A–D , BMP-2 and LIF enhance the expression of RARα, which is necessary for retinoic acid-induced GFAP synthesis. On the other hand, retinoic acid ( R ) has no effect on the key phosphorylation

    Article Snippet: For immunoprecipitations, antibodies against the following proteins were used at the indicated dilutions: p300/CBP and RARα (1:20, Santa Cruz Biotechnology) and Stat3 and Smad1 (1:100 and 1:200, Cell Signaling Technologies, Danvers, MA).

    Techniques: Expressing

    The crbp1 gene transfection reverses L5 effects on STRA6 cascades and renal cell injury. A: Western blots show LOX1, STRA6, CRBP1, RARα, RXRα, pJNK, pSmad2, and collagen 1 (Col1) expression in cell lysate of pCMV6-transfected and pCMV6-crbp1-transfected HK-2 cells under PBS (Ctl), L1, or L5 treatment for 24 h (n = 3). Quantitative analysis showed a significant difference for the increase of LOX1/actin (B); the decrease of STRA6/actin (C), CRBP1/actin (D), RARα/actin (E), and RXRα/actin (F); the increase of pJNK/JNK (G), pSmad2/Smad2 (H), caspase 3/actin (I), and collagen 1/actin (J) under L5 stimulation in pCMV6-transfected cells. These changes caused by L5 treatment were reversed in pCMV6-crbp1-transfected cells. K: ELISA showed that pCMV6-crbp1-transfection reversed the elevation of TGFβ 1 concentration under L5 treatment in the culture medium of HK-2 cells. L: Cytochemistry images show immunoreactive staining of STRA6 (green fluorescence) was recovered by pCMV6-crbp1-transfection in L5-treated HK-2 cells (crbp1+L5). All results are represented as mean ± SE. § P

    Journal: Journal of Lipid Research

    Article Title: Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved [S]

    doi: 10.1194/jlr.M067215

    Figure Lengend Snippet: The crbp1 gene transfection reverses L5 effects on STRA6 cascades and renal cell injury. A: Western blots show LOX1, STRA6, CRBP1, RARα, RXRα, pJNK, pSmad2, and collagen 1 (Col1) expression in cell lysate of pCMV6-transfected and pCMV6-crbp1-transfected HK-2 cells under PBS (Ctl), L1, or L5 treatment for 24 h (n = 3). Quantitative analysis showed a significant difference for the increase of LOX1/actin (B); the decrease of STRA6/actin (C), CRBP1/actin (D), RARα/actin (E), and RXRα/actin (F); the increase of pJNK/JNK (G), pSmad2/Smad2 (H), caspase 3/actin (I), and collagen 1/actin (J) under L5 stimulation in pCMV6-transfected cells. These changes caused by L5 treatment were reversed in pCMV6-crbp1-transfected cells. K: ELISA showed that pCMV6-crbp1-transfection reversed the elevation of TGFβ 1 concentration under L5 treatment in the culture medium of HK-2 cells. L: Cytochemistry images show immunoreactive staining of STRA6 (green fluorescence) was recovered by pCMV6-crbp1-transfection in L5-treated HK-2 cells (crbp1+L5). All results are represented as mean ± SE. § P

    Article Snippet: Materials Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ1 (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA).

    Techniques: Transfection, Western Blot, Expressing, CTL Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Fluorescence

    L5 decreases STRA6 cascades and injures the kidney. The protein of kidney samples (n = 3) was extracted from saline-injected (Ctl), L1-injected (L1), and L5-injected (L5) mice, as well as L5-injected LOX1 −/− mice (LOX1 −/− +L5) after injection for 4 weeks. A: Western blots showed that L5 reduced STRA6, CRBP1, RARα, RARγ, and RXRα, but increased LOX1, pJNK, p-p38MAPK (p-p38), TGFβ 1 , pSmad2, caspase 3 (Casp3), and collagen 1 (Col1) in L5-injected mice. In LOX1 −/− mice, these changes caused by L5 were diminished. B: Bar graphs show that LOX1 increased and STRA6, CRBP1, RARα, RARγ, and RXRα decreased in L5-injected mice. These changes caused by L5 were attenuated in L5-injected LOX1 −/− mice. C: Bar graphs indicate that pJNK/JNK, p-p38/p38, and pSmad2/Smad2 ratios increased in L5-injected mice. These changes caused by L5 were attenuated in L5-injected LOX1 −/− mice. D: TGFβ 1 , caspase 3, and collagen 1 levels of L5-injected mice increased. These changes caused by L5 were recovered in L5-injected LOX1 −/− mice. E: RT-PCR analysis shows that L5 decreased PCR products of STRA6, CRBP1, RARα, and RXRα in L5-injected mice, but there was no effect in L5-injected LOX1 −/− mice. All results are represented as mean ± SE; * P

    Journal: Journal of Lipid Research

    Article Title: Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved [S]

    doi: 10.1194/jlr.M067215

    Figure Lengend Snippet: L5 decreases STRA6 cascades and injures the kidney. The protein of kidney samples (n = 3) was extracted from saline-injected (Ctl), L1-injected (L1), and L5-injected (L5) mice, as well as L5-injected LOX1 −/− mice (LOX1 −/− +L5) after injection for 4 weeks. A: Western blots showed that L5 reduced STRA6, CRBP1, RARα, RARγ, and RXRα, but increased LOX1, pJNK, p-p38MAPK (p-p38), TGFβ 1 , pSmad2, caspase 3 (Casp3), and collagen 1 (Col1) in L5-injected mice. In LOX1 −/− mice, these changes caused by L5 were diminished. B: Bar graphs show that LOX1 increased and STRA6, CRBP1, RARα, RARγ, and RXRα decreased in L5-injected mice. These changes caused by L5 were attenuated in L5-injected LOX1 −/− mice. C: Bar graphs indicate that pJNK/JNK, p-p38/p38, and pSmad2/Smad2 ratios increased in L5-injected mice. These changes caused by L5 were attenuated in L5-injected LOX1 −/− mice. D: TGFβ 1 , caspase 3, and collagen 1 levels of L5-injected mice increased. These changes caused by L5 were recovered in L5-injected LOX1 −/− mice. E: RT-PCR analysis shows that L5 decreased PCR products of STRA6, CRBP1, RARα, and RXRα in L5-injected mice, but there was no effect in L5-injected LOX1 −/− mice. All results are represented as mean ± SE; * P

    Article Snippet: Materials Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ1 (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA).

    Techniques: Injection, CTL Assay, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    L5 suppresses STRA6 cascades and increases cell injury in HK-2 cells. A: Western blots show LOX1, STRA6, CRBP1, RARα, RARγ, RXRα, pJNK, pSmad2, and collagen 1 (Col1) in control siRNA (siC)-transfected or LOX1 siRNA (siLOX1)-transfected HK-2 cells (n = 3) after PBS (Ctl), L1, or L5 treatment for 24 h. In L5-treated HK-2 cells, quantitative analysis showed that LOX1 (B) protein levels significantly increased; STRA6 (C), CRBP1 (D), RARα (E), RARγ (F), and RXRα (G) protein levels decreased; pJNK/JNK (H) and pSmad2/Smad2 (I) ratios increased; and collagen 1 (J) increased. These changes were reversed in LOX1 siRNA-transfected L5-injected HK-2 cells, as compared with L5-injected siC mice. K: Representative images (400×) show immunoreactive staining for STRA6 (red fluorescence) emerged to cell membrane staining (green fluorescence) on chamber slides of Ctl-, L1-, and L5-treated cells, and L5-treated LOX1 siRNA-transfected cells. L: ELISA showed an increase of TGFβ 1 concentration in L5-treated siC cells, but not in L5-treated siLOX1 cells. M: Cytochemistry images show the nucleus of apoptotic cells (green fluorescence) emerged to staining of nucleus (blue fluorescence) in Ctl-, L1-, and L5-treated cells, and in L5-treated LOX1 siRNA-transfected cells. N: Number of apoptotic cells significantly increased in L5-treated siC cells, but not in L5-treated siLOX1 cells. O: Real-time PCR analysis showed that LOX1 siRNA significantly increased mRNA levels of STRA6, CRBP1, RARα, and RXRα in L5-treated HK-2 cells. All results are represented as mean ± SE. * P

    Journal: Journal of Lipid Research

    Article Title: Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved [S]

    doi: 10.1194/jlr.M067215

    Figure Lengend Snippet: L5 suppresses STRA6 cascades and increases cell injury in HK-2 cells. A: Western blots show LOX1, STRA6, CRBP1, RARα, RARγ, RXRα, pJNK, pSmad2, and collagen 1 (Col1) in control siRNA (siC)-transfected or LOX1 siRNA (siLOX1)-transfected HK-2 cells (n = 3) after PBS (Ctl), L1, or L5 treatment for 24 h. In L5-treated HK-2 cells, quantitative analysis showed that LOX1 (B) protein levels significantly increased; STRA6 (C), CRBP1 (D), RARα (E), RARγ (F), and RXRα (G) protein levels decreased; pJNK/JNK (H) and pSmad2/Smad2 (I) ratios increased; and collagen 1 (J) increased. These changes were reversed in LOX1 siRNA-transfected L5-injected HK-2 cells, as compared with L5-injected siC mice. K: Representative images (400×) show immunoreactive staining for STRA6 (red fluorescence) emerged to cell membrane staining (green fluorescence) on chamber slides of Ctl-, L1-, and L5-treated cells, and L5-treated LOX1 siRNA-transfected cells. L: ELISA showed an increase of TGFβ 1 concentration in L5-treated siC cells, but not in L5-treated siLOX1 cells. M: Cytochemistry images show the nucleus of apoptotic cells (green fluorescence) emerged to staining of nucleus (blue fluorescence) in Ctl-, L1-, and L5-treated cells, and in L5-treated LOX1 siRNA-transfected cells. N: Number of apoptotic cells significantly increased in L5-treated siC cells, but not in L5-treated siLOX1 cells. O: Real-time PCR analysis showed that LOX1 siRNA significantly increased mRNA levels of STRA6, CRBP1, RARα, and RXRα in L5-treated HK-2 cells. All results are represented as mean ± SE. * P

    Article Snippet: Materials Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ1 (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA).

    Techniques: Western Blot, Transfection, CTL Assay, Injection, Mouse Assay, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction

    JNK inhibitor represses L5 effect on STRA6 cascades and renal injury. A: Western blots show STRA6, CRBP1, RARα, RARγ, RXRα, pSmad2, and collagen 1 (Col1) of Ctl, L1-, or L5-treated RETCs with or without SP600125 treatment for 24 h (n = 3). Quantitative analysis showed that L5 treatment decreased STRA6 (B), CRBP1 (C), RARα (D), RARγ (E), and RXRα (F), but increased the pSmad2/Smad2 ratio (G) and collagen 1 (H). All of these effects of L5 were reversed in SP600125-treated RETCs under L5-stimulation. I: Representative optical microscopy images show nuclei of apoptotic cells (thick dark stain) in Ctl, L1-treated, and L5-treated cells with or without SP600125 treatment. SP600125 decreased the nuclei of apoptotic cells in RETCs under L5-stimulation. J: ELISA showed that SP600125 treatment attenuated the increase of TGFβ 1 concentration in the culture medium of L5-treated RETCs. K: Real-time PCR analysis revealed that SP600125 significantly recovered the decreased mRNA levels of STRA6, CRBP1, RARα, and RXRα in L5-treated RETCs. All results are represented as mean ± SE. * P

    Journal: Journal of Lipid Research

    Article Title: Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved [S]

    doi: 10.1194/jlr.M067215

    Figure Lengend Snippet: JNK inhibitor represses L5 effect on STRA6 cascades and renal injury. A: Western blots show STRA6, CRBP1, RARα, RARγ, RXRα, pSmad2, and collagen 1 (Col1) of Ctl, L1-, or L5-treated RETCs with or without SP600125 treatment for 24 h (n = 3). Quantitative analysis showed that L5 treatment decreased STRA6 (B), CRBP1 (C), RARα (D), RARγ (E), and RXRα (F), but increased the pSmad2/Smad2 ratio (G) and collagen 1 (H). All of these effects of L5 were reversed in SP600125-treated RETCs under L5-stimulation. I: Representative optical microscopy images show nuclei of apoptotic cells (thick dark stain) in Ctl, L1-treated, and L5-treated cells with or without SP600125 treatment. SP600125 decreased the nuclei of apoptotic cells in RETCs under L5-stimulation. J: ELISA showed that SP600125 treatment attenuated the increase of TGFβ 1 concentration in the culture medium of L5-treated RETCs. K: Real-time PCR analysis revealed that SP600125 significantly recovered the decreased mRNA levels of STRA6, CRBP1, RARα, and RXRα in L5-treated RETCs. All results are represented as mean ± SE. * P

    Article Snippet: Materials Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ1 (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA).

    Techniques: Western Blot, CTL Assay, Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction

    STRA6 siRNA damages STRA6 cascades and induces renal cell injury. A: Western blots show LOX1, STRA6, CRBP1, RARα, RXRα, pJNK, pSmad2, and collagen 1 (Col1) in cell lysate of STRA6 siRNA (siSTRA6) or control siRNA (siC) transfected HK-2 cells cultured in PBS (Ctl)-, L1-, and L5-treated HK-2 cells for 24 h (n = 3). Quantitative analysis showed significance for the increase of LOX1/actin (B); the decrease of STRA6/actin (C), CRBP1/actin (D), RARα/actin (E), RXRα/actin (F), pJNK/JNK (G), pSmad2/Smad2 (H), and collagen 1/actin (I) in STRA6 siRNA-transfected HK-2 cells in Ctl and L1 groups. J: ELISA also showed a significant increase of TGFβ 1 in culture medium of siSTRA6-transfected HK-2 cells in Ctl, L1, and L5 groups. K: Cytochemistry images show that STRA6 siRNA transfection increased the nuclei of apoptotic cells (green fluorescence) in Ctl- and L1-treated cells. All results are represented as mean ± SE. § P

    Journal: Journal of Lipid Research

    Article Title: Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved [S]

    doi: 10.1194/jlr.M067215

    Figure Lengend Snippet: STRA6 siRNA damages STRA6 cascades and induces renal cell injury. A: Western blots show LOX1, STRA6, CRBP1, RARα, RXRα, pJNK, pSmad2, and collagen 1 (Col1) in cell lysate of STRA6 siRNA (siSTRA6) or control siRNA (siC) transfected HK-2 cells cultured in PBS (Ctl)-, L1-, and L5-treated HK-2 cells for 24 h (n = 3). Quantitative analysis showed significance for the increase of LOX1/actin (B); the decrease of STRA6/actin (C), CRBP1/actin (D), RARα/actin (E), RXRα/actin (F), pJNK/JNK (G), pSmad2/Smad2 (H), and collagen 1/actin (I) in STRA6 siRNA-transfected HK-2 cells in Ctl and L1 groups. J: ELISA also showed a significant increase of TGFβ 1 in culture medium of siSTRA6-transfected HK-2 cells in Ctl, L1, and L5 groups. K: Cytochemistry images show that STRA6 siRNA transfection increased the nuclei of apoptotic cells (green fluorescence) in Ctl- and L1-treated cells. All results are represented as mean ± SE. § P

    Article Snippet: Materials Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ1 (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA).

    Techniques: Western Blot, Transfection, Cell Culture, CTL Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

    A functional RARE is located in exon 1a of the human EVI1 gene. (A) Schematic of the genomic region at the 5′-end of the human EVI1 gene and of the fragments cloned into pGL3basic, and luciferase reporter assays. Stippled line, EVI1 upstream region; grey boxes, exons (exons 1b and 1c are too small to be depicted in this schematic); solid lines, introns. The positions of cloned fragments are indicated relative to the transcriptional start site of EVI1_1a (GenBank accession no. BX640908). pRARE-tk-luc contains two copies of the human RARβ RARE and the tk minimal promoter in pGL2, and was used as a positive control for the ATRA response. For luciferase assays, the indicated reporter plasmids were transfected into NTERA-2 cells, together with the renilla luciferase plasmid pRL; 10 μ m ATRA or an equivalent amount of dimethylsulfoxide was added 1 day after transfection. Another ∼ 24 h later, cells were lysed and luciferase activities were measured. Relative luciferase units (RLU) were derived by normalizing firefly to renilla luciferase activities. Error bars represent the standard deviations between duplicate measurements. White bars, dimethylsulfoxide; black bars, ATRA. (B) Schematic of EVI1(+15/+1106)/pGL3 and its derivative constructs, and luciferase reporter assays. Grey box, exon 1a; black boxes, predicted RAREs. In EVI1(+86/+1106)mut/pGL3, the six nucelotides comprising the second half-site of the EVI1 RARE were mutated (indicated by a cross). Luciferase reporter gene assays were performed as in (A). (C) RT-PCR confirming that the RARE of the EVI1 gene is located within its transcribed region. NTERA-2 cells were incubated with ATRA for 0 or 72 h. RNA was extracted, treated with DNaseI, reverse transcribed and amplified using primers flanking the EVI1 RARE (EVI1_RARE-F, EVI1_RARE-R; Table S1B ), an intron-spanning primer pair located in a more proximal region of EVI1_1a (EVI1_1a-fwd, EVI1_1a-rev; Table S1A ) and a primer pair for the housekeeping gene cyclophilinD (cycD-F, cycD-R; Table S1A ). The effectiveness of the DNaseI treatment was verified with primers for the microsatellite marker D22S686 (D22S686-F, D22S686-R; Table S1B ). (D) ChIP was performed on NTERA-2 cells treated with ATRA or dimethylsulfoxide for 24 h using an antibody specific to RARα, a pan-RXR antibody, or unspecific rabbit IgG as a negative control. Immunoprecipitated chromatin and input DNA as a positive control were amplified with primers flanking the EVI1 RARE (EVI1_RARE-F, EVI1_RARE-R; Table S1B ), primers for the RARβ RARE (RARβ_RARE-F, RARβ_RARE-R; Table S1B ) or with negative control primers located in EVI1 exon 3L (EVI1_ex3L-F; EVI1_ex3L-R; Table S1B ).

    Journal: The Febs Journal

    Article Title: Zinc finger transcription factor ecotropic viral integration site 1 is induced by all-trans retinoic acid (ATRA) and acts as a dual modulator of the ATRA response

    doi: 10.1111/j.1742-4658.2009.07398.x

    Figure Lengend Snippet: A functional RARE is located in exon 1a of the human EVI1 gene. (A) Schematic of the genomic region at the 5′-end of the human EVI1 gene and of the fragments cloned into pGL3basic, and luciferase reporter assays. Stippled line, EVI1 upstream region; grey boxes, exons (exons 1b and 1c are too small to be depicted in this schematic); solid lines, introns. The positions of cloned fragments are indicated relative to the transcriptional start site of EVI1_1a (GenBank accession no. BX640908). pRARE-tk-luc contains two copies of the human RARβ RARE and the tk minimal promoter in pGL2, and was used as a positive control for the ATRA response. For luciferase assays, the indicated reporter plasmids were transfected into NTERA-2 cells, together with the renilla luciferase plasmid pRL; 10 μ m ATRA or an equivalent amount of dimethylsulfoxide was added 1 day after transfection. Another ∼ 24 h later, cells were lysed and luciferase activities were measured. Relative luciferase units (RLU) were derived by normalizing firefly to renilla luciferase activities. Error bars represent the standard deviations between duplicate measurements. White bars, dimethylsulfoxide; black bars, ATRA. (B) Schematic of EVI1(+15/+1106)/pGL3 and its derivative constructs, and luciferase reporter assays. Grey box, exon 1a; black boxes, predicted RAREs. In EVI1(+86/+1106)mut/pGL3, the six nucelotides comprising the second half-site of the EVI1 RARE were mutated (indicated by a cross). Luciferase reporter gene assays were performed as in (A). (C) RT-PCR confirming that the RARE of the EVI1 gene is located within its transcribed region. NTERA-2 cells were incubated with ATRA for 0 or 72 h. RNA was extracted, treated with DNaseI, reverse transcribed and amplified using primers flanking the EVI1 RARE (EVI1_RARE-F, EVI1_RARE-R; Table S1B ), an intron-spanning primer pair located in a more proximal region of EVI1_1a (EVI1_1a-fwd, EVI1_1a-rev; Table S1A ) and a primer pair for the housekeeping gene cyclophilinD (cycD-F, cycD-R; Table S1A ). The effectiveness of the DNaseI treatment was verified with primers for the microsatellite marker D22S686 (D22S686-F, D22S686-R; Table S1B ). (D) ChIP was performed on NTERA-2 cells treated with ATRA or dimethylsulfoxide for 24 h using an antibody specific to RARα, a pan-RXR antibody, or unspecific rabbit IgG as a negative control. Immunoprecipitated chromatin and input DNA as a positive control were amplified with primers flanking the EVI1 RARE (EVI1_RARE-F, EVI1_RARE-R; Table S1B ), primers for the RARβ RARE (RARβ_RARE-F, RARβ_RARE-R; Table S1B ) or with negative control primers located in EVI1 exon 3L (EVI1_ex3L-F; EVI1_ex3L-R; Table S1B ).

    Article Snippet: Chromatin was diluted 1 : 10 into ChIP buffer (OneDay ChIP kit, Diagenode, Liege, Belgium) and agitated overnight at 4 °C with 8 μg of RARα antibody (sc-551, Santa Cruz, CA, USA), pan-RXR antibody (sc-774, Santa Cruz) or rabbit IgG.

    Techniques: Functional Assay, Clone Assay, Luciferase, Positive Control, Transfection, Plasmid Preparation, Derivative Assay, Construct, Reverse Transcription Polymerase Chain Reaction, Incubation, Amplification, Marker, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation

    PPARβ/δ suppresses MMP2 activity by interfering with RAR signaling A. A putative RAR RE in the human MMP2 promoter. +1 represented the transcriptional start site. B. Representative EMSA of nuclear extracts from NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells were incubated with either double-stranded oligonucleotides (oligo) encoding the RAR RE MMP2 promoter or mutated oligonucleotides (mutant oligo). Black arrowheads indicated the presence of oligonucleotide-protein complexes. White arrowheads indicated the super shift of oligonucleotide-protein-anti-RAR-antibody complexes. C. Representative photomicrograph of the ChIP assay for RAR occupancy on the MMP2 promoter. D. RARα protein expression in NT2/D1, MigR1 and hPPARβ/δ cells transiently transfected with pSG5-RARα plasmid. E, F. CYP26A1 and MMP2 mRNA expression in MigR1 and hPPARβ/δ cells or cells transiently over-expressing RARα after atRA treatment, respectively. G. Left panel, activities of MMP2 and MMP9 in MigR1 and hPPARβ/δ cells transiently over-expressing RAR after atRA treatment, right panel, relative activity of pro-MMP2 in MigR1 and hPPARβ/δ cells. H. ChIP-qPCR showing effect of PPARβ/δ on RARα occupancy on MMP2 promoter in cells over-expressing PPARβ/δ or RARα following atRA and/or GW0742 treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Journal: Oncotarget

    Article Title: Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

    doi:

    Figure Lengend Snippet: PPARβ/δ suppresses MMP2 activity by interfering with RAR signaling A. A putative RAR RE in the human MMP2 promoter. +1 represented the transcriptional start site. B. Representative EMSA of nuclear extracts from NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells were incubated with either double-stranded oligonucleotides (oligo) encoding the RAR RE MMP2 promoter or mutated oligonucleotides (mutant oligo). Black arrowheads indicated the presence of oligonucleotide-protein complexes. White arrowheads indicated the super shift of oligonucleotide-protein-anti-RAR-antibody complexes. C. Representative photomicrograph of the ChIP assay for RAR occupancy on the MMP2 promoter. D. RARα protein expression in NT2/D1, MigR1 and hPPARβ/δ cells transiently transfected with pSG5-RARα plasmid. E, F. CYP26A1 and MMP2 mRNA expression in MigR1 and hPPARβ/δ cells or cells transiently over-expressing RARα after atRA treatment, respectively. G. Left panel, activities of MMP2 and MMP9 in MigR1 and hPPARβ/δ cells transiently over-expressing RAR after atRA treatment, right panel, relative activity of pro-MMP2 in MigR1 and hPPARβ/δ cells. H. ChIP-qPCR showing effect of PPARβ/δ on RARα occupancy on MMP2 promoter in cells over-expressing PPARβ/δ or RARα following atRA and/or GW0742 treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Article Snippet: Antibodies against RARα, progesterone receptor (PR), and normal rabbit-IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were individually added to histone-DNA complexes for immunoprecipitation of protein bound chromatin.

    Techniques: Activity Assay, Incubation, Mutagenesis, Chromatin Immunoprecipitation, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    PPARβ/δ interferes with atRA-stimulated signaling in NT2/D1 cells A. Quantitative western blot analysis of RXRα, RARα, OCT3/4, FABP5 and CRABPII expression in NT2/D1, NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells. B, C. Quantitative western blot analysis of RXRα, RARα, CYP26A1, FABP5 and CRABPII expression in NT2/D1, MigR1 and hPPARβ/δ cells in response to atRA treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Journal: Oncotarget

    Article Title: Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

    doi:

    Figure Lengend Snippet: PPARβ/δ interferes with atRA-stimulated signaling in NT2/D1 cells A. Quantitative western blot analysis of RXRα, RARα, OCT3/4, FABP5 and CRABPII expression in NT2/D1, NT2/D1-MigR1 (MigR1) and NT2/D1-hPPARβ/δ (hPPARβ/δ) cells. B, C. Quantitative western blot analysis of RXRα, RARα, CYP26A1, FABP5 and CRABPII expression in NT2/D1, MigR1 and hPPARβ/δ cells in response to atRA treatment. Values represent mean ± S.E.M. Values with different superscript letters are significantly different at p ≤ 0.05.

    Article Snippet: Antibodies against RARα, progesterone receptor (PR), and normal rabbit-IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were individually added to histone-DNA complexes for immunoprecipitation of protein bound chromatin.

    Techniques: Western Blot, Expressing

    The level of expression of PCNA affects transcriptional activation by RARα. ( A ) HeLa cells were transfected with a retinoid-inducible reporter gene, RARα, RXRα, and/or PCNA expression vectors. The luciferase activity was assayed after treatment with 1 μM atRA overnight. RARα, PCNA and actin levels were assayed by western blot (lower panel). ( B ) Overexpressed PCNA is hyperacetylated. HeLa cells were transfected either with an expression vector coding for PCNA or chloramphenicol acetyltransferase as a control. Forty-eight hours later, nuclear extracts were prepared and incubated with a mouse IgG (control) or with an anti-PCNA antibody. Immune complexes were immunoprecipitated with protein G–Sepharose beads and analyzed by western blot using an anti-PCNA antibody (upper panel) or an anti-acetylated lysine antibody. The left panel corresponds to one tenth of the input in the immunoprecipitation assay. ( C ) PCNA knockdown increases atRA responsiveness in HeLa cells. HeLa cells were transfected with a retinoid-inducible reporter gene, RARα and RXRα expression vector after a 24 h treatment with anti-PCNA siRNA or control siRNA. atRA treatment and luciferase assays were as in (A). ( D ) PCNA knockdown increases atRA responsiveness in the presence of physiological levels of hRARα. MCF7 cells were treated with control or anti-PCNA siRNA and transfected with a retinoid-inducible reporter gene, p800-tkLuc (see Materials and Methods). atRA responsiveness of the system was monitored as above. ( E ) Effect of PCNA concentration on atRA responsiveness of endogenous genes. HeLa cells were treated as above with control or anti-PCNA siRNA, and mRNA levels were assessed by semi-quantitative RT–PCR after an overnight challenge with 1 μM atRA (RNAs). A representative experiment is shown; similar results were obtained twice in independent experiments. PCNA and actin levels were assayed by western blot (proteins).

    Journal: Nucleic Acids Research

    Article Title: The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein-protein interaction

    doi: 10.1093/nar/gki745

    Figure Lengend Snippet: The level of expression of PCNA affects transcriptional activation by RARα. ( A ) HeLa cells were transfected with a retinoid-inducible reporter gene, RARα, RXRα, and/or PCNA expression vectors. The luciferase activity was assayed after treatment with 1 μM atRA overnight. RARα, PCNA and actin levels were assayed by western blot (lower panel). ( B ) Overexpressed PCNA is hyperacetylated. HeLa cells were transfected either with an expression vector coding for PCNA or chloramphenicol acetyltransferase as a control. Forty-eight hours later, nuclear extracts were prepared and incubated with a mouse IgG (control) or with an anti-PCNA antibody. Immune complexes were immunoprecipitated with protein G–Sepharose beads and analyzed by western blot using an anti-PCNA antibody (upper panel) or an anti-acetylated lysine antibody. The left panel corresponds to one tenth of the input in the immunoprecipitation assay. ( C ) PCNA knockdown increases atRA responsiveness in HeLa cells. HeLa cells were transfected with a retinoid-inducible reporter gene, RARα and RXRα expression vector after a 24 h treatment with anti-PCNA siRNA or control siRNA. atRA treatment and luciferase assays were as in (A). ( D ) PCNA knockdown increases atRA responsiveness in the presence of physiological levels of hRARα. MCF7 cells were treated with control or anti-PCNA siRNA and transfected with a retinoid-inducible reporter gene, p800-tkLuc (see Materials and Methods). atRA responsiveness of the system was monitored as above. ( E ) Effect of PCNA concentration on atRA responsiveness of endogenous genes. HeLa cells were treated as above with control or anti-PCNA siRNA, and mRNA levels were assessed by semi-quantitative RT–PCR after an overnight challenge with 1 μM atRA (RNAs). A representative experiment is shown; similar results were obtained twice in independent experiments. PCNA and actin levels were assayed by western blot (proteins).

    Article Snippet: The precleared lysate was incubated overnight at 4°C with 2 μg of anti-RARα antiserum (sc-551; Santa Cruz Biotechnology) or non-specific IgG.

    Techniques: Expressing, Activation Assay, Transfection, Luciferase, Activity Assay, Western Blot, Plasmid Preparation, Incubation, Immunoprecipitation, Concentration Assay, Quantitative RT-PCR

    Domain mapping of the RARα–PCNA interaction. ( A ) The RARα DBD is sufficient for RAR–PCNA interaction. Full-length RARα, or the isolated DBD or LBD fused to GST were incubated with labeled PCNA and incubated with GST alone or GST-RAR bound to Sepharose beads. After extensive washing, bound material was analyzed by SDS–PAGE and bound PCNA was visualized by autoradiography. ( B ) GST pull-down assay with full-length PCNA. The indicated RAR mutants were translated and labeled in vitro and incubated with a Sepharose–glutathione/GST–PCNA slurry. The interaction was analyzed as in (A). A similar assay was carried out with RXRα. ( C ) Mapping of the RAR interaction region within PCNA. Various PCNA deletion mutants were labeled and incubated with the full-length RARα-GST fusion protein. Bound PCNA was analyzed as above. ( D ) Minimal PCNA–RAR interaction domain. A Sepharose-bound GST fused to amino acids 1–61 from PCNA was incubated with labeled, full-length RARα. Bound material was assayed as above.

    Journal: Nucleic Acids Research

    Article Title: The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein-protein interaction

    doi: 10.1093/nar/gki745

    Figure Lengend Snippet: Domain mapping of the RARα–PCNA interaction. ( A ) The RARα DBD is sufficient for RAR–PCNA interaction. Full-length RARα, or the isolated DBD or LBD fused to GST were incubated with labeled PCNA and incubated with GST alone or GST-RAR bound to Sepharose beads. After extensive washing, bound material was analyzed by SDS–PAGE and bound PCNA was visualized by autoradiography. ( B ) GST pull-down assay with full-length PCNA. The indicated RAR mutants were translated and labeled in vitro and incubated with a Sepharose–glutathione/GST–PCNA slurry. The interaction was analyzed as in (A). A similar assay was carried out with RXRα. ( C ) Mapping of the RAR interaction region within PCNA. Various PCNA deletion mutants were labeled and incubated with the full-length RARα-GST fusion protein. Bound PCNA was analyzed as above. ( D ) Minimal PCNA–RAR interaction domain. A Sepharose-bound GST fused to amino acids 1–61 from PCNA was incubated with labeled, full-length RARα. Bound material was assayed as above.

    Article Snippet: The precleared lysate was incubated overnight at 4°C with 2 μg of anti-RARα antiserum (sc-551; Santa Cruz Biotechnology) or non-specific IgG.

    Techniques: Isolation, Incubation, Labeling, SDS Page, Autoradiography, Pull Down Assay, In Vitro

    A yeast two-hybrid screen identify PCNA as a partner of human RARα. ( A ) Main features of the PCNA molecule. Domains of PCNA and some of its most prominent partners are indicated. ( B ) The AF2-inactivated RAR mutant (RAR2K) interacts with PCNA in a ligand-independent manner. Yeasts were transformed with various pLex12 expression vectors (empty vector, Ras fusion, RAR2K fusion) and baits fused to the Gal4 activation domain (empty vector, Raf, RXR DBD, PCNA). The β-galactosidase activity resulting from the activation of the LacZ gene hooked to a LexA response element was detected 24 h after retinoic acid challenge. The Ras/Raf system was used as a positive control. ( C ) PCNA overexpression inhibits retinoid-induced transcription. HeLa cells were cotransfected in 6-wells cluster (∼1.5 × 10 5 cells) with a retinoid-inducible reporter gene pGL3(RARE) 3 tkLuc (500 ng) designated as DR5tk-Luc hereafter, or a tk-driven luciferase reporter gene (500 ng, tk-Luc, see Materials and Methods), RARα and RXRα expression vectors (50 ng of each), and a 10-fold stoichiometric excess of a PCNA expression vector. Twenty-four hours after transfection, cells were challenged, or not, overnight with 1 μM atRA and luciferase activity was assayed. Results are expressed as the mean of three independent experiments performed in triplicate. ( D ) Preventing coactivator binding site occupancy does not block PCNA repressive effect. HeLa cells were transfected as above with a reporter gene, RARα and RXRα expression vectors, with or without PCNA (1 μg) and increasing amounts (0.25, 0.5 and 1 μg) of an expression vector coding for the NR box of DRIP205 (LXXLL) or its mutated inactive version (LXXAA). Luciferase activity was assayed as described above.

    Journal: Nucleic Acids Research

    Article Title: The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein-protein interaction

    doi: 10.1093/nar/gki745

    Figure Lengend Snippet: A yeast two-hybrid screen identify PCNA as a partner of human RARα. ( A ) Main features of the PCNA molecule. Domains of PCNA and some of its most prominent partners are indicated. ( B ) The AF2-inactivated RAR mutant (RAR2K) interacts with PCNA in a ligand-independent manner. Yeasts were transformed with various pLex12 expression vectors (empty vector, Ras fusion, RAR2K fusion) and baits fused to the Gal4 activation domain (empty vector, Raf, RXR DBD, PCNA). The β-galactosidase activity resulting from the activation of the LacZ gene hooked to a LexA response element was detected 24 h after retinoic acid challenge. The Ras/Raf system was used as a positive control. ( C ) PCNA overexpression inhibits retinoid-induced transcription. HeLa cells were cotransfected in 6-wells cluster (∼1.5 × 10 5 cells) with a retinoid-inducible reporter gene pGL3(RARE) 3 tkLuc (500 ng) designated as DR5tk-Luc hereafter, or a tk-driven luciferase reporter gene (500 ng, tk-Luc, see Materials and Methods), RARα and RXRα expression vectors (50 ng of each), and a 10-fold stoichiometric excess of a PCNA expression vector. Twenty-four hours after transfection, cells were challenged, or not, overnight with 1 μM atRA and luciferase activity was assayed. Results are expressed as the mean of three independent experiments performed in triplicate. ( D ) Preventing coactivator binding site occupancy does not block PCNA repressive effect. HeLa cells were transfected as above with a reporter gene, RARα and RXRα expression vectors, with or without PCNA (1 μg) and increasing amounts (0.25, 0.5 and 1 μg) of an expression vector coding for the NR box of DRIP205 (LXXLL) or its mutated inactive version (LXXAA). Luciferase activity was assayed as described above.

    Article Snippet: The precleared lysate was incubated overnight at 4°C with 2 μg of anti-RARα antiserum (sc-551; Santa Cruz Biotechnology) or non-specific IgG.

    Techniques: Two Hybrid Screening, Mutagenesis, Transformation Assay, Expressing, Plasmid Preparation, Activation Assay, Activity Assay, Positive Control, Over Expression, Luciferase, Transfection, Binding Assay, Blocking Assay

    PCNA interacts with RARα in intact cells and in vitro . ( A ) PCNA coimmunoprecipitates with RARα in HeLa cells. HeLa cells were transfected with a RARα expression vector and total extracts were immunoprecipitated with Sepharose-conjugated anti-RAR or PCNA antibodies (or mouse IgG as a control), respectively, after retinoid challenge. Immune complexes were analyzed by western blots for the presence of RARα and PCNA. ( B ) PCNA coimmunoprecipitates with RARα in MCF7 cells. MCF7 total extracts were immunoprecipitated and analyzed as above. ( C ) The RXR–RAR heterodimer interacts with PCNA in vitro . RXR and/or RAR were labeled by in vitro translation and incubated with a Glutathione–Sepharose matrix coupled to either GST–PCNA or the receptor interacting domain of SRC1 fused to GST or GST alone. When indicated, a DR5 RARE was added to the binding mixture. Receptors interacting with the matrix were visualized by autoradiography. ( D ) PCNA associates with a retinoid-induced promoter in intact cells. HeLa cells were cotransfected with PCNA, RXR and RAR expression vectors together with DR5tk-Luc retinoid-inducible reporter gene. A ChIP assay was carried out with a mouse IgG (control), an anti-RARα, an anti-SRC1 or an anti-PCNA antibody. The precipitated DNA was amplified by PCR and analyzed on an agarose gel. Naive P19 cells were used for a similar assay to quantify the amount of endogenous RARβ2 promoter DNA associated to PCNA.

    Journal: Nucleic Acids Research

    Article Title: The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein-protein interaction

    doi: 10.1093/nar/gki745

    Figure Lengend Snippet: PCNA interacts with RARα in intact cells and in vitro . ( A ) PCNA coimmunoprecipitates with RARα in HeLa cells. HeLa cells were transfected with a RARα expression vector and total extracts were immunoprecipitated with Sepharose-conjugated anti-RAR or PCNA antibodies (or mouse IgG as a control), respectively, after retinoid challenge. Immune complexes were analyzed by western blots for the presence of RARα and PCNA. ( B ) PCNA coimmunoprecipitates with RARα in MCF7 cells. MCF7 total extracts were immunoprecipitated and analyzed as above. ( C ) The RXR–RAR heterodimer interacts with PCNA in vitro . RXR and/or RAR were labeled by in vitro translation and incubated with a Glutathione–Sepharose matrix coupled to either GST–PCNA or the receptor interacting domain of SRC1 fused to GST or GST alone. When indicated, a DR5 RARE was added to the binding mixture. Receptors interacting with the matrix were visualized by autoradiography. ( D ) PCNA associates with a retinoid-induced promoter in intact cells. HeLa cells were cotransfected with PCNA, RXR and RAR expression vectors together with DR5tk-Luc retinoid-inducible reporter gene. A ChIP assay was carried out with a mouse IgG (control), an anti-RARα, an anti-SRC1 or an anti-PCNA antibody. The precipitated DNA was amplified by PCR and analyzed on an agarose gel. Naive P19 cells were used for a similar assay to quantify the amount of endogenous RARβ2 promoter DNA associated to PCNA.

    Article Snippet: The precleared lysate was incubated overnight at 4°C with 2 μg of anti-RARα antiserum (sc-551; Santa Cruz Biotechnology) or non-specific IgG.

    Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Labeling, Incubation, Binding Assay, Autoradiography, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing RARα or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.

    Journal: The Journal of Neuroscience

    Article Title: Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling

    doi: 10.1523/JNEUROSCI.2121-16.2017

    Figure Lengend Snippet: A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing RARα or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.

    Article Snippet: Protein-DNA complexes were incubated with the following antibodies overnight at 4°C on a rotator: normal rabbit IgG (Millipore catalog #12–371 RRID:AB-145840, 2 μg), rabbit anti-GCN5L2 (Cell Signaling Technology catalog #3305S RRID:AB-2128281, 1:200), rabbit anti-RARα (Santa Cruz Biotechnology catalog #sc-551 RRID:AB-2177750, 1:200), rabbit anti-TACC1 (Sigma-Aldrich catalog #SAB4502910 RRID:AB-10747337, 1:100).

    Techniques: Binding Assay, Expressing, Inhibition, Activity Assay

    A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing RARα or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.

    Journal: The Journal of Neuroscience

    Article Title: Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling

    doi: 10.1523/JNEUROSCI.2121-16.2017

    Figure Lengend Snippet: A proposed model for GCN5-mediated regulation of diencephalic size. In the absence of RA, a poised transcriptional complex containing RARα or RARγ, GCN5/SAGA, and TACC1 localizes to the RAREs of some RA-responsive genes, with TACC1 acting as a negative regulator of transcription in the absence of ligand. Upon RA binding to the RAR, GCN5-mediated acetylation of TACC1 causes dissociation of TACC1 from the complex, allowing transcription of downstream RA target genes. RA signaling in the dorsal p3 and p2 domains of the developing diencephalon inhibits WNT signaling, which is a positive regulator of Shh expression in the alar plate through its inhibition of Gli3 expression. Loss of GCN5 activity results in decreased RA signaling, which causes widespread upregulation of WNT and SHH through increased repression of Gli3 expression and expansion of the ZLI.

    Article Snippet: Protein interactions were assayed via Western blot using the following antibodies: rabbit anti-GCN5L2 (Cell Signaling Technology catalog #3305S RRID:AB-2128281, 1:1000), goat anti-RARα (Santa Cruz Biotechnology catalog #sc-15040 RRID:AB-2177747, 1:50), rabbit anti-TACC1 (Sigma-Aldrich catalog #SAB4502910 RRID:AB-10747337, 1:500), and mouse anti-β-tubulin (Sigma-Aldrich catalog #T4026 RRID:AB-477577, 1:2000).

    Techniques: Binding Assay, Expressing, Inhibition, Activity Assay

    Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.

    Journal: Scientific Reports

    Article Title: SP1 and RARα regulate AGAP2 expression in cancer

    doi: 10.1038/s41598-018-36888-x

    Figure Lengend Snippet: Mechanism of ATRA-mediated AGAP2 transcription. ( a ) Sheared chromatin of DU145 cells grown under AGAP2 expression conditions was used for immunoprecipitation using 2 μg of antibody (a rabbit IgG as negative control, a rabbit antibody against RNApol II as positive control, a rabbit anti-RARα, a rabbit anti-RXRα or a rabbit anti-PCAF antibody) and optimised primers were used to amplify a region specific to the AGAP2 ). Data are represented as fold enrichment: fold enrich in signal relative to the IgG background signal. ( b ) Diagram representing the proposed mechanism of ATRA-mediated activation of AGAP2 transcription. In the presence of ATRA (lower panel), the heterodimer RARα/RXRα would recruit the lysine acetyl transferase PCAF to activate transcription and the recruitment of SP1 would also be enhanced.

    Article Snippet: For immunoprecipitations, 2 µg of anti-RARα goat antibody (L-15; Santa Cruz Biotechnology) were coupled with the 50 µl of Surebeads protein – G (BioRad) in a total volume of 200 µl of TBST for 10 minutes at room temperature.

    Techniques: Expressing, Immunoprecipitation, Negative Control, Positive Control, Activation Assay

    In vitro differentiation of myeloid progenitors from mCG PML-RARα cells with or without neutrophil elastase. (Left) Mean cell numbers and standard deviations over time in culture. Data represent cultures of bone marrow from one of two independent

    Journal: Molecular and Cellular Biology

    Article Title: Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor ? Activities in Early Myeloid Cells

    doi: 10.1128/MCB.25.1.23-33.2005

    Figure Lengend Snippet: In vitro differentiation of myeloid progenitors from mCG PML-RARα cells with or without neutrophil elastase. (Left) Mean cell numbers and standard deviations over time in culture. Data represent cultures of bone marrow from one of two independent

    Article Snippet: Western blot analysis for PML-RARα protein with an anti-human RARα antibody (C-20; Santa Cruz) was performed as previously described ( ).

    Techniques: In Vitro

    Cell death induced by transient expression of PML-RARα. (A) GFP fusion proteins were visualized by fluorescence microscopy 8 h after transfection in U937 (top) or K562 (bottom) cells. Staining with an antibody recognizing human PML is also shown,

    Journal: Molecular and Cellular Biology

    Article Title: Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor ? Activities in Early Myeloid Cells

    doi: 10.1128/MCB.25.1.23-33.2005

    Figure Lengend Snippet: Cell death induced by transient expression of PML-RARα. (A) GFP fusion proteins were visualized by fluorescence microscopy 8 h after transfection in U937 (top) or K562 (bottom) cells. Staining with an antibody recognizing human PML is also shown,

    Article Snippet: Western blot analysis for PML-RARα protein with an anti-human RARα antibody (C-20; Santa Cruz) was performed as previously described ( ).

    Techniques: Expressing, Fluorescence, Microscopy, Transfection, Staining

    Neutrophil elastase levels and PML-RARα protein expression in cell line models of APL. (A) Western blotting with a human RARα-specific antibody for U937 cells, untreated U937-PR9 cells, U937-PR9 cells induced with 100 μM ZnSO 4

    Journal: Molecular and Cellular Biology

    Article Title: Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor ? Activities in Early Myeloid Cells

    doi: 10.1128/MCB.25.1.23-33.2005

    Figure Lengend Snippet: Neutrophil elastase levels and PML-RARα protein expression in cell line models of APL. (A) Western blotting with a human RARα-specific antibody for U937 cells, untreated U937-PR9 cells, U937-PR9 cells induced with 100 μM ZnSO 4

    Article Snippet: Western blot analysis for PML-RARα protein with an anti-human RARα antibody (C-20; Santa Cruz) was performed as previously described ( ).

    Techniques: Expressing, Western Blot

    Relationship between PML-RARα toxicity and its cleavage by neutrophil elastase. (A) Clonogenic data for U937 cells transfected with the indicated GFP fusion cDNAs. WT, wild-type PML-RARα; 2VR, = PML-RARα containing the

    Journal: Molecular and Cellular Biology

    Article Title: Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor ? Activities in Early Myeloid Cells

    doi: 10.1128/MCB.25.1.23-33.2005

    Figure Lengend Snippet: Relationship between PML-RARα toxicity and its cleavage by neutrophil elastase. (A) Clonogenic data for U937 cells transfected with the indicated GFP fusion cDNAs. WT, wild-type PML-RARα; 2VR, = PML-RARα containing the

    Article Snippet: Western blot analysis for PML-RARα protein with an anti-human RARα antibody (C-20; Santa Cruz) was performed as previously described ( ).

    Techniques: Transfection

    Model for the roles of PML-RARα dose and NE expression in APL penetrance. At the time when differentiating myeloid cells reach the stage of peak cathepsin G transgene activation (green cells), the population experiences a normal (stochastic) distribution

    Journal: Molecular and Cellular Biology

    Article Title: Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor ? Activities in Early Myeloid Cells

    doi: 10.1128/MCB.25.1.23-33.2005

    Figure Lengend Snippet: Model for the roles of PML-RARα dose and NE expression in APL penetrance. At the time when differentiating myeloid cells reach the stage of peak cathepsin G transgene activation (green cells), the population experiences a normal (stochastic) distribution

    Article Snippet: Western blot analysis for PML-RARα protein with an anti-human RARα antibody (C-20; Santa Cruz) was performed as previously described ( ).

    Techniques: Expressing, Activation Assay

    RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.

    Journal: The Journal of Biological Chemistry

    Article Title: Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells *

    doi: 10.1074/jbc.M901779200

    Figure Lengend Snippet: RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.

    Article Snippet: For determining the specific RARα-binding region in Sp1, both in vitro translated RARα (10 μl) and wt/truncated HA-Sp1 (10 μl) proteins were incubated in TNE buffer (250 m m NaCl, 5 m m EDTA, 10 m m Tris-HCl, pH 7.4, and 1× protease inhibitor) in a total volume of 250 μl on ice for 3 h, and immunoprecipitated with rabbit polyclonal anti-RARα antibody (sc-551, Santa Cruz Biotechnology) at the final concentration of 10 μg/ml at 4 °C for 4 h on a rotating mixer.

    Techniques: Recombinant, Incubation, Immunoprecipitation, Western Blot, Negative Control, Co-Immunoprecipitation Assay, In Vitro, Binding Assay

    RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.

    Journal: The Journal of Biological Chemistry

    Article Title: Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells *

    doi: 10.1074/jbc.M901779200

    Figure Lengend Snippet: RARα interacts with Sp1 via zinc finger domains in Sp1. A , RARα but not RXRα interacts with Sp1. 200 ng of human recombinant Sp1 protein was incubated with either 200 ng of GST-RARα protein or 200 ng of GST-RXRα protein in 50 μl of PBS on ice for 2 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody ( lane 4 ) or rabbit polyclonal anti-RXRα antibody ( lane 5 ) at 4 °C overnight. With the addition of Protein A beads, samples were incubated at 4 °C for another 2 h. After extensive washes, beads were boiled in 2× SDS sample buffer for 5 min followed by Western blot with mouse monoclonal anti-Sp1 antibody. Incubations of Sp1 protein with anti-RARα antibody ( lane 6 ) or anti-RXRα antibody ( lane 7 ) only were used as a negative control for IP. Pure GST-RARα and GST-RXRα proteins as input used in co-IP assay was verified by Western blot with anti-RARα ( lane 8 ) and anti-RXRα ( lane 9 ) antibodies, respectively. All inputs shown were 100% input. B , both in vitro translated RARα (10 μl) and wt/truncated HA-tagged Sp1 (10 μl) proteins were incubated in TNE buffer on ice for 3 h followed by immunoprecipitation with rabbit polyclonal anti-RARα antibody at 4 °C for 4 h and incubation with Protein A beads at 4 °C for another 1 h. Interactions of RARα with various wt/truncated Sp1 were analyzed by Western blot with mouse monoclonal anti-HA antibody and TrueBlot HRP anti-mouse IgG (secondary antibody). The incubation of in vitro translated wt HA-Sp1 with anti-RARα antibody only was used as a negative control ( NTC ) for IP. C , Western blot analysis of 20% input fraction used in co-IP assay ( B ) with anti-HA and anti-RARα antibodies. D , a schematic representation of wt/truncated HA-tagged Sp1 protein structures and their RARα-binding abilities.

    Article Snippet: Monoclonal anti-RXRα (sc-46659), anti-Sp1 (sc-17824), anti-β-actin (sc-47778), anti-GST (sc-138), polyclonal anti-RARα (sc-773), anti-RARα (sc-551), anti-RXRα (sc-774), and anti-GFP (sc-8334) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Recombinant, Incubation, Immunoprecipitation, Western Blot, Negative Control, Co-Immunoprecipitation Assay, In Vitro, Binding Assay

    Model for RARα dependent epigenetic regulation. ( A ) Ligand-independent binding of RARα/RXRα heterodimers to the Mest promoter region is required to maintain transcriptionally permissive histone modifications (H3K4me3 and H3K9/K14ac) and relatively low methylation levels of promoter CpG islands (upper panel). Knockout of RARα results in higher levels of Mest promoter methylation, loss of permissive histone modifications, and gain of repressive histone modifications (lower panel). ( B ) Actively transcribed genes display relative low levels of promoter methylation and high levels of H3K9/K14ac and H3K4me3 (Mest and Tex13 in Wt, Stmn2 and Slc38a4 in RARα −/− ), whereas silenced genes display high levels of promoter methylation and, if paternally expressed, high H3K9me3 levels (Slc38a4 in Wt, Mest in RARα −/− ). Transcriptional start sites (TSS) and relative transcriptional activities are indicated by the arrows and arrow sizes, respectively. The relative levels of CpG promoter methylation are denoted by black lollipops (the CpG-rich regions assessed each span the TSS but for clarity are drawn upstream of the TSS).

    Journal: Nucleic Acids Research

    Article Title: Epigenetic regulation by RAR? maintains ligand-independent transcriptional activity

    doi: 10.1093/nar/gkr637

    Figure Lengend Snippet: Model for RARα dependent epigenetic regulation. ( A ) Ligand-independent binding of RARα/RXRα heterodimers to the Mest promoter region is required to maintain transcriptionally permissive histone modifications (H3K4me3 and H3K9/K14ac) and relatively low methylation levels of promoter CpG islands (upper panel). Knockout of RARα results in higher levels of Mest promoter methylation, loss of permissive histone modifications, and gain of repressive histone modifications (lower panel). ( B ) Actively transcribed genes display relative low levels of promoter methylation and high levels of H3K9/K14ac and H3K4me3 (Mest and Tex13 in Wt, Stmn2 and Slc38a4 in RARα −/− ), whereas silenced genes display high levels of promoter methylation and, if paternally expressed, high H3K9me3 levels (Slc38a4 in Wt, Mest in RARα −/− ). Transcriptional start sites (TSS) and relative transcriptional activities are indicated by the arrows and arrow sizes, respectively. The relative levels of CpG promoter methylation are denoted by black lollipops (the CpG-rich regions assessed each span the TSS but for clarity are drawn upstream of the TSS).

    Article Snippet: Western blots The SDS–PAGE and western blot analysis were performed as described ( , ) using RARα primary Ab (Cat. #sc-551, Santa Cruz, CA, USA) in a 1:1000 dilution and HPR-conjugated anti-rabbit secondary Ab (Cat. #sc-2030, Santa Cruz, CA, USA) in a 1:10 000 dilution.

    Techniques: Binding Assay, Methylation, Knock-Out

    RARα and RXRα binding to target promoters. Promoter specific ChIP were quantified by real-time PCR on chromatin IPed from Wt and from RARα −/− cells treated with vehicle or RA (vehicle–0 h; gray and RA–24 h; dark gray bars). The chromatin was IPed using ( A ) an RARα Ab, ( B ) an RXRα Ab or ( C ) IgG negative control. The western blot in Figure 1 f demonstrates antibody specificity toward RARα. The average signal from IgG IP was set to 1 (marked by the light gray background in A, B and C). The data represent four independent IPs for each Ab, harvesting new chromatin for each IP. Statistical significance is demonstrated by P

    Journal: Nucleic Acids Research

    Article Title: Epigenetic regulation by RAR? maintains ligand-independent transcriptional activity

    doi: 10.1093/nar/gkr637

    Figure Lengend Snippet: RARα and RXRα binding to target promoters. Promoter specific ChIP were quantified by real-time PCR on chromatin IPed from Wt and from RARα −/− cells treated with vehicle or RA (vehicle–0 h; gray and RA–24 h; dark gray bars). The chromatin was IPed using ( A ) an RARα Ab, ( B ) an RXRα Ab or ( C ) IgG negative control. The western blot in Figure 1 f demonstrates antibody specificity toward RARα. The average signal from IgG IP was set to 1 (marked by the light gray background in A, B and C). The data represent four independent IPs for each Ab, harvesting new chromatin for each IP. Statistical significance is demonstrated by P

    Article Snippet: Western blots The SDS–PAGE and western blot analysis were performed as described ( , ) using RARα primary Ab (Cat. #sc-551, Santa Cruz, CA, USA) in a 1:1000 dilution and HPR-conjugated anti-rabbit secondary Ab (Cat. #sc-2030, Santa Cruz, CA, USA) in a 1:10 000 dilution.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Western Blot

    The Epigenetic Signatures of Mest, Tex13, Stmn2 and Slc38a4 promoter regions. ( A ) Mest and Tex13 promoters displayed increased methylation in RARα −/− relative to Wt cells. In contrast, Slc38a4 and Stmn2 displayed decreased methylation in RARα −/− relative to Wt cells. Each horizontal line represents the methylation status of an independent allele. The numbers below the figures indicate the CpG position relative to the transcriptional start site (+1). ( B–D ) Promoter specific ChIP were quantified by real-time PCR on chromatin IPed from Wt and from RARα knockout cells treated with vehicle or RA (vehicle—0 h; gray and RA—24 h; dark gray bars). (B) Histone modification in F9 Wt and RARα −/− cells. H3K9/14ac modifications (upper panel). In RARα −/− cells H3K9/K14ac levels at the Mest and Tex13 promoters is decreased, while levels at the Stmn2 and Slc38a4 promoters is increased relative to Wt. (C) H3K9me3 modifications (middle panel). In RARα −/− cells the H3K9me3 level at the Mest promoter is increased, while the level at the Slc38a4 promoter is decreased. H3K9me3 levels at the Tex13 and Stmn2 promoters are not significantly changed in RARα −/− cells. A low signal (15- to 30-fold above the IgG) for H3K9me3 is seen at the Cyp26a1 promoter. (D) H3K4me3 modifications (lower panel). In RARα −/− cells H3K4me3 levels at the Mest and Tex13 promoters are decreased, whereas the levels at the Stmn2 and Slc38a4 promoters are increased relative to Wt. The signal from the IgG IP was set to 1 for each PCR. The data represent three independent IPs for each Ab, harvesting new chromatin for each IP. Statistical significance is demonstrated by P- values below 0.05 for the indicated comparisons. (C) Mest proximal promoter region. The promoter (excluding the sequences in italics) was evaluated for CpG methylation (bold). Underlined sequences indicate putative elements: RARE (DR1), NFkB-binding site, TATA box (TATA), transcriptional start site (TSS) and the exact region targeted by ChIP primers (labels to the right).

    Journal: Nucleic Acids Research

    Article Title: Epigenetic regulation by RAR? maintains ligand-independent transcriptional activity

    doi: 10.1093/nar/gkr637

    Figure Lengend Snippet: The Epigenetic Signatures of Mest, Tex13, Stmn2 and Slc38a4 promoter regions. ( A ) Mest and Tex13 promoters displayed increased methylation in RARα −/− relative to Wt cells. In contrast, Slc38a4 and Stmn2 displayed decreased methylation in RARα −/− relative to Wt cells. Each horizontal line represents the methylation status of an independent allele. The numbers below the figures indicate the CpG position relative to the transcriptional start site (+1). ( B–D ) Promoter specific ChIP were quantified by real-time PCR on chromatin IPed from Wt and from RARα knockout cells treated with vehicle or RA (vehicle—0 h; gray and RA—24 h; dark gray bars). (B) Histone modification in F9 Wt and RARα −/− cells. H3K9/14ac modifications (upper panel). In RARα −/− cells H3K9/K14ac levels at the Mest and Tex13 promoters is decreased, while levels at the Stmn2 and Slc38a4 promoters is increased relative to Wt. (C) H3K9me3 modifications (middle panel). In RARα −/− cells the H3K9me3 level at the Mest promoter is increased, while the level at the Slc38a4 promoter is decreased. H3K9me3 levels at the Tex13 and Stmn2 promoters are not significantly changed in RARα −/− cells. A low signal (15- to 30-fold above the IgG) for H3K9me3 is seen at the Cyp26a1 promoter. (D) H3K4me3 modifications (lower panel). In RARα −/− cells H3K4me3 levels at the Mest and Tex13 promoters are decreased, whereas the levels at the Stmn2 and Slc38a4 promoters are increased relative to Wt. The signal from the IgG IP was set to 1 for each PCR. The data represent three independent IPs for each Ab, harvesting new chromatin for each IP. Statistical significance is demonstrated by P- values below 0.05 for the indicated comparisons. (C) Mest proximal promoter region. The promoter (excluding the sequences in italics) was evaluated for CpG methylation (bold). Underlined sequences indicate putative elements: RARE (DR1), NFkB-binding site, TATA box (TATA), transcriptional start site (TSS) and the exact region targeted by ChIP primers (labels to the right).

    Article Snippet: Western blots The SDS–PAGE and western blot analysis were performed as described ( , ) using RARα primary Ab (Cat. #sc-551, Santa Cruz, CA, USA) in a 1:1000 dilution and HPR-conjugated anti-rabbit secondary Ab (Cat. #sc-2030, Santa Cruz, CA, USA) in a 1:10 000 dilution.

    Techniques: Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Knock-Out, Modification, Polymerase Chain Reaction, CpG Methylation Assay, Binding Assay

    Ectopic RARα expression affects Mest transcript levels in F9 cells. ( A ) Full-length RARα 2 was stably expressed in F9 RARα −/− cells (upper right). Transcript levels assessed by real-time PCR demonstrate partial restoration of Mest transcript levels (upper left), but no reversal of the overall promoter methylation (lower left). Stmn2 transcript levels were not affected (middle panel). Tex13 and Slc38a4 transcript levels were inconsistent between the two independent RARα 2 restoration lines (lower middle and right panel). The P- values show a comparison of RARα 2 restoration cell lines to the RARα knockout cell line. The data represent three independent assays, harvesting new RNA for each experiment. ( B ) The PML–RARα oncogene was stably expressed in F9 Wt cells. Transcript levels assessed by real-time PCR (upper left panel) suggest a dominant negative function of the PML–RARα protein for Mest associated with increased levels of promoter methylation (lower left panel), while Tex13, Slc38a4 and Stmn2 transcript levels were not affected (Wt, 24 h RA was set to 1). RA induced transcription of Cyp26a1 was impaired in PML–RARα expressing cells (upper right panel). The duration of the RA treatment is indicated by the bar color (0 h; gray, 8 h; light gray, and 24 h; dark gray bars). The P- values show a comparison of the PML–RARα cell line to the F9 Wt cells. The data represent three independent assays, harvesting new RNA for each experiment.

    Journal: Nucleic Acids Research

    Article Title: Epigenetic regulation by RAR? maintains ligand-independent transcriptional activity

    doi: 10.1093/nar/gkr637

    Figure Lengend Snippet: Ectopic RARα expression affects Mest transcript levels in F9 cells. ( A ) Full-length RARα 2 was stably expressed in F9 RARα −/− cells (upper right). Transcript levels assessed by real-time PCR demonstrate partial restoration of Mest transcript levels (upper left), but no reversal of the overall promoter methylation (lower left). Stmn2 transcript levels were not affected (middle panel). Tex13 and Slc38a4 transcript levels were inconsistent between the two independent RARα 2 restoration lines (lower middle and right panel). The P- values show a comparison of RARα 2 restoration cell lines to the RARα knockout cell line. The data represent three independent assays, harvesting new RNA for each experiment. ( B ) The PML–RARα oncogene was stably expressed in F9 Wt cells. Transcript levels assessed by real-time PCR (upper left panel) suggest a dominant negative function of the PML–RARα protein for Mest associated with increased levels of promoter methylation (lower left panel), while Tex13, Slc38a4 and Stmn2 transcript levels were not affected (Wt, 24 h RA was set to 1). RA induced transcription of Cyp26a1 was impaired in PML–RARα expressing cells (upper right panel). The duration of the RA treatment is indicated by the bar color (0 h; gray, 8 h; light gray, and 24 h; dark gray bars). The P- values show a comparison of the PML–RARα cell line to the F9 Wt cells. The data represent three independent assays, harvesting new RNA for each experiment.

    Article Snippet: Western blots The SDS–PAGE and western blot analysis were performed as described ( , ) using RARα primary Ab (Cat. #sc-551, Santa Cruz, CA, USA) in a 1:1000 dilution and HPR-conjugated anti-rabbit secondary Ab (Cat. #sc-2030, Santa Cruz, CA, USA) in a 1:10 000 dilution.

    Techniques: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Methylation, Knock-Out, Dominant Negative Mutation

    Gene expression analyses of wild-type and RARα knockout cells. Relative transcript levels were identified by microarray analysis and the genes differentially expressed (2-fold or more difference in transcript levels between wild-type and RARα knockout cells) were plotted as fold difference in presence of RA against the fold difference in vehicle-treated cells ( A right panel, transcript levels in RARα −/− cells > 2-fold transcript levels in Wt cells; C left panel, transcript levels in RARα −/− cells

    Journal: Nucleic Acids Research

    Article Title: Epigenetic regulation by RAR? maintains ligand-independent transcriptional activity

    doi: 10.1093/nar/gkr637

    Figure Lengend Snippet: Gene expression analyses of wild-type and RARα knockout cells. Relative transcript levels were identified by microarray analysis and the genes differentially expressed (2-fold or more difference in transcript levels between wild-type and RARα knockout cells) were plotted as fold difference in presence of RA against the fold difference in vehicle-treated cells ( A right panel, transcript levels in RARα −/− cells > 2-fold transcript levels in Wt cells; C left panel, transcript levels in RARα −/− cells

    Article Snippet: Western blots The SDS–PAGE and western blot analysis were performed as described ( , ) using RARα primary Ab (Cat. #sc-551, Santa Cruz, CA, USA) in a 1:1000 dilution and HPR-conjugated anti-rabbit secondary Ab (Cat. #sc-2030, Santa Cruz, CA, USA) in a 1:10 000 dilution.

    Techniques: Expressing, Knock-Out, Microarray

    Effects of arsenicals on differentiation of NB4 cells and PML-RARα fusion protein degradation NB4 cells were exposed to 1 μM of arsenicals for 24 and 72 h to determine the changes in PRAM-1 protein A. and quantified by Image J public domain software B. Additionally, NB4 cells were stained with Wright-Giemsa C. and the expression of CD11b in NB4 cells were determined by flow cytometry D. Degradation of PML-RARα fusion protein in NB4 cells were determined by western blot with RARα antibody E. For immunofluorescence, NB4 cells were double-labeled with PML (red) and SUMO-1 (green) after exposure to 1 μM arsenicals for 12 h. Blue fluorescence indicates cell nucleus. Cells were imaged with a laser scanning confocal microscope F. ATRA was used as positive control in the present study. Asterisks indicate a significant difference from the untreated control group at * p

    Journal: Oncotarget

    Article Title: Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    doi:

    Figure Lengend Snippet: Effects of arsenicals on differentiation of NB4 cells and PML-RARα fusion protein degradation NB4 cells were exposed to 1 μM of arsenicals for 24 and 72 h to determine the changes in PRAM-1 protein A. and quantified by Image J public domain software B. Additionally, NB4 cells were stained with Wright-Giemsa C. and the expression of CD11b in NB4 cells were determined by flow cytometry D. Degradation of PML-RARα fusion protein in NB4 cells were determined by western blot with RARα antibody E. For immunofluorescence, NB4 cells were double-labeled with PML (red) and SUMO-1 (green) after exposure to 1 μM arsenicals for 12 h. Blue fluorescence indicates cell nucleus. Cells were imaged with a laser scanning confocal microscope F. ATRA was used as positive control in the present study. Asterisks indicate a significant difference from the untreated control group at * p

    Article Snippet: Antibodies Primary antibodies RARα, PRAM-1, rabbit anti-Poly (ADP-ribose) polymerase (PARP) polyclonal antibody, Cyt c, JNK, P-JNK, P-PERK, eif2α, rabbit anti-human PML, anti-human SUMO-1, Ub Antibody were purchased from Santa Cruz Biotechnology (CA, USA).

    Techniques: Software, Staining, Expressing, Flow Cytometry, Cytometry, Western Blot, Immunofluorescence, Labeling, Fluorescence, Microscopy, Positive Control

    Examination of PML-NBs formation or PML and PML-RARα proteins degradation in HEK293T and HeLa cells after exposure to arsenicals Degradation of PML-RARα fusion protein was examined in PML-RARα -transfected HeLa cells following exposure to 4 μM of arsenicals at indicated time points A. Likewise, degradation of PML protein was determined in PML -HEK293T cells following exposure to 4 μM of arsenicals for 6 h B. Formation of PML-NBs in PML or PML-RARα overexpressed HEK293T C. or HeLa cells D. were determined after exposure to arsenicals for 6 h. Cells were double-labeled with PML (green) and SUMO-1 (red). Blue fluorescence indicates cell nucleus. Arrow indicates the PML-NBs.

    Journal: Oncotarget

    Article Title: Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    doi:

    Figure Lengend Snippet: Examination of PML-NBs formation or PML and PML-RARα proteins degradation in HEK293T and HeLa cells after exposure to arsenicals Degradation of PML-RARα fusion protein was examined in PML-RARα -transfected HeLa cells following exposure to 4 μM of arsenicals at indicated time points A. Likewise, degradation of PML protein was determined in PML -HEK293T cells following exposure to 4 μM of arsenicals for 6 h B. Formation of PML-NBs in PML or PML-RARα overexpressed HEK293T C. or HeLa cells D. were determined after exposure to arsenicals for 6 h. Cells were double-labeled with PML (green) and SUMO-1 (red). Blue fluorescence indicates cell nucleus. Arrow indicates the PML-NBs.

    Article Snippet: Antibodies Primary antibodies RARα, PRAM-1, rabbit anti-Poly (ADP-ribose) polymerase (PARP) polyclonal antibody, Cyt c, JNK, P-JNK, P-PERK, eif2α, rabbit anti-human PML, anti-human SUMO-1, Ub Antibody were purchased from Santa Cruz Biotechnology (CA, USA).

    Techniques: Transfection, Labeling, Fluorescence

    miR-126 is not regulated by flow-eliciting RARα/RXRα or HDAC-3/5/7 signaling in ECs. ( A ) ECs were kept under static condition or exposed to flows over the 24-h test period, and their miR-126 expression was determined by qPCR. ( B and C

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: miR-126 is not regulated by flow-eliciting RARα/RXRα or HDAC-3/5/7 signaling in ECs. ( A ) ECs were kept under static condition or exposed to flows over the 24-h test period, and their miR-126 expression was determined by qPCR. ( B and C

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction

    PS induces sustained increases in the expressions, nuclear accumulations, and association of RARα and RXRα, which modulate miR-10a and downstream GATA6/VCAM-1 expressions in ECs. ECs were kept under static (C) or shear condition. ( A and

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: PS induces sustained increases in the expressions, nuclear accumulations, and association of RARα and RXRα, which modulate miR-10a and downstream GATA6/VCAM-1 expressions in ECs. ECs were kept under static (C) or shear condition. ( A and

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques:

    KLF-2 is involved in flow-mediated EC miR-10a expression. ECs were transfected with specific siRNA of RARα, RXRα ( A ), KLF-2, or WT KLF-2 ( B–D ) and then exposed to static or shear conditions. As controls, cells were transfected

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: KLF-2 is involved in flow-mediated EC miR-10a expression. ECs were transfected with specific siRNA of RARα, RXRα ( A ), KLF-2, or WT KLF-2 ( B–D ) and then exposed to static or shear conditions. As controls, cells were transfected

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Flow Cytometry, Expressing, Transfection

    RXRα and HDAC-3/5/7 serve as “enhancer” and “repressor” to modulate flow-regulated RARα-RARE binding in ECs. ECs were kept under static (C) or flow condition ( A ). ECs were transfected with control siRNA

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: RXRα and HDAC-3/5/7 serve as “enhancer” and “repressor” to modulate flow-regulated RARα-RARE binding in ECs. ECs were kept under static (C) or flow condition ( A ). ECs were transfected with control siRNA

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Flow Cytometry, Binding Assay, Transfection

    Expression of RARα, RXRα, miR-10a, GATA6, and VCAM-1 is flow pattern-specific in the native circulation. ( A–F ) The inner and outer curvatures of the AA and the straight segment of the TA ( A ) of normal rats ( n = 5) were examined

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: Expression of RARα, RXRα, miR-10a, GATA6, and VCAM-1 is flow pattern-specific in the native circulation. ( A–F ) The inner and outer curvatures of the AA and the straight segment of the TA ( A ) of normal rats ( n = 5) were examined

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Expressing, Flow Cytometry

    Statistical analysis of PS-induced expressions and nuclear accumulations of RARα and RXRα, which modulate miR-10a and downstream GATA6/VCAM-1 expression in ECs. ECs were kept under static condition or exposed to shear condition. ( A and

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: Statistical analysis of PS-induced expressions and nuclear accumulations of RARα and RXRα, which modulate miR-10a and downstream GATA6/VCAM-1 expression in ECs. ECs were kept under static condition or exposed to shear condition. ( A and

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Expressing

    Transfection efficiencies for all of the siRNA experiments in ECs. ECs were transfected with control siRNA or specific siRNA of RARα, RXRα, HDAC3, HDAC5, or HDAC7 at various concentrations for 48 h. The expressions of target molecules

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: Transfection efficiencies for all of the siRNA experiments in ECs. ECs were transfected with control siRNA or specific siRNA of RARα, RXRα, HDAC3, HDAC5, or HDAC7 at various concentrations for 48 h. The expressions of target molecules

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Transfection

    OS inhibits miR-10a expression through RARα-HDAC-3/5/7 associations, with the up-regulations of GATA6 and VCAM-1 in ECs. ( A – C ) ECs were kept under static (C) or shear condition. ECs were transfected with control siRNA or HDAC-specific

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

    doi: 10.1073/pnas.1621425114

    Figure Lengend Snippet: OS inhibits miR-10a expression through RARα-HDAC-3/5/7 associations, with the up-regulations of GATA6 and VCAM-1 in ECs. ( A – C ) ECs were kept under static (C) or shear condition. ECs were transfected with control siRNA or HDAC-specific

    Article Snippet: In brief, after exposure to OS or PS for 4 h, cells were fixed with 4% (wt/vol) paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 20 min, and then incubated with rabbit anti-human RARα antibody (sc-551; Santa Cruz Biotechnology) and mouse anti-human RXRα antibody (sc-46659; Santa Cruz Biotechnology) at 37 °C for 1 h. After three washes in PBS, the cells were labeled with PLUS oligonucleotide-conjugated anti-mouse antibody (Duolink II, Olink Bioscience) and MINUS oligonucleotide-conjugated anti-rabbit antibody (Duolink II, Olink Bioscience) at 37 °C for 1 h. After the addition of template oligonucleotide, annealing, and ligation, the circularized template was amplified via polymerase, and the amplified sequence was detected by hybridization with a Texas Red-labeled probe.

    Techniques: Expressing, Transfection

    pRB colocalizes with PML within nuclear bodies and is delocalized by PML-RARα expression. (A) Immunofluorescence experiments using an anti-pRB monoclonal antibody (α-RB; aRB1C1) revealed with a rhodamine-conjugated anti-mouse antibody and an anti-PML polyclonal antibody (α-PML; 2912A) revealed with an FITC-conjugated anti-rabbit antibody were performed with IB4 and U937 cells. Superimposition of PML and pRB staining is shown in the righthand panels, indicating colocalization of PML and pRB within NBs in both cell lines. (B) Similar experiments were performed with the U937 PR9 clone before and after induction of PML-RARα expression with ZnSO 4 . Superimposition of PML and pRB staining is shown in the righthand panels, revealing that pRB is delocalized into the PML-RARα microspeckles by the expression of the fusion protein. The anti-RB antibody aRB1C1 does not cross-react with PML-RARα, as revealed by Western blotting experiments and immunoprecipitations of in vitro-translated PML-RARα polypeptides (our unpublished results).

    Journal: Molecular and Cellular Biology

    Article Title: The Promyelocytic Leukemia Gene Product (PML) Forms Stable Complexes with the Retinoblastoma Protein

    doi:

    Figure Lengend Snippet: pRB colocalizes with PML within nuclear bodies and is delocalized by PML-RARα expression. (A) Immunofluorescence experiments using an anti-pRB monoclonal antibody (α-RB; aRB1C1) revealed with a rhodamine-conjugated anti-mouse antibody and an anti-PML polyclonal antibody (α-PML; 2912A) revealed with an FITC-conjugated anti-rabbit antibody were performed with IB4 and U937 cells. Superimposition of PML and pRB staining is shown in the righthand panels, indicating colocalization of PML and pRB within NBs in both cell lines. (B) Similar experiments were performed with the U937 PR9 clone before and after induction of PML-RARα expression with ZnSO 4 . Superimposition of PML and pRB staining is shown in the righthand panels, revealing that pRB is delocalized into the PML-RARα microspeckles by the expression of the fusion protein. The anti-RB antibody aRB1C1 does not cross-react with PML-RARα, as revealed by Western blotting experiments and immunoprecipitations of in vitro-translated PML-RARα polypeptides (our unpublished results).

    Article Snippet: PML-RARα was immunoprecipitated either with monoclonal antibody PG-M3 or with anti-RARα polyclonal antibody C-20 (Santa Cruz); Western blotting analysis was performed with the anti-RARα-F antibody (gift from P. Chambon).

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot, In Vitro