anti sp1 Cell Signaling Technology Inc Search Results


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  • 97
    Cell Signaling Technology Inc anti sp1
    Schematic model of BMX regulates VEGFR2 transcription by interaction with <t>Sp1.</t> The SH2/SH3 domain of BMX confers its nuclear localization in ECs. Nuclear BMX in an active form interacts with (and possibly phosphorylates) Sp1 to facilitate the recruitment of Sp1 to the VEGFR2 promoter and its transcription in ECs
    Anti Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti sp1
    siRNA -mediated depletion of <t>SP1</t> or c-JUN expression in ER+ EC cells abolished TAM-stimulated promoter activity of TFF3 and growth in Matrigel culture (A) TFF3 promoter activity and (B) growth in Matrigel of EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. Depletion of SP1 or c-JUN expression was achieved using transient-transfection of si - RNA directed to SP1 or c-JUN transcript respectively, as described in materials and methods. Universal controls (scrambled oligo) were used for transfection control as described in materials and methods. The luciferase assay was performed as described in materials and methods. FM were 10%FBS, standard media conditions as per ATCC propagation instructions; and CSF-PRFM were charcoal striped 10% FBS, phenol-red free media. Statistical significance was assessed by using an unpaired two-tailed Student's t test ( P
    Rabbit Anti Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 6 article reviews
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    88
    Cell Signaling Technology Inc mouse anti sp1
    EMSA of (A) I <t>Sp1</t> (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.
    Mouse Anti Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc monoclonal rabbit anti human sp1
    Western Blot analysis of β-catenin expression in RMS tumor cell lines. (A) Western Blot analysis of ß-catenin expression in various RMS tumor cell lines. ß-actin served as loading control. (B) Western Blot analysis of ß-catenin expression in three different RMS tumor cell lines after 48 h treatment with either FH535, XAV939, ß-catenin siRNA, or (C) WNT3A treatment. ß-actin served as loading control. (D) Densitometric quantification of the Western Blots shown in (C) using ImageJ. Expression of ß-catenin after treatment (+) was normalized to ß-actin and compared to untreated cells (−). (E) subcellular localization of ß-catenin in the RMS tumor cell lines with and without WNT3A stimulation. GAPDH served as cytoplasmatic and <t>SP1</t> as nuclear marker to check purity of the protein isolation from different compartments. CP, cytoplasmatic; N, nuclear.
    Monoclonal Rabbit Anti Human Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc sp1 d4c3
    Western blot analysis of <t>Sp1</t> protein in 2DG-treated NCCIT cells. The whole cell lysates of NCCIT cells treated with 2DG for the indicated times (0, 24, 72, or 168 h) were precipitated with an anti-Sp1 antibody (D4C3), and the precipitated proteins were immunoblotted with D4C3, anti- O -GlcNAc antibodies (RL2 or CTD110.6), anti-Phosphoserine/threonine (P-Ser/Thr), anti-SUMO1, or anti-Ubiquitin. These transcriptional modifications were observed in Sp1 proteins [6] . Among these modifications, only O -GlcNAcylation was clearly detected in the D4C3 precipitates. The Sp1 levels in whole cell lysates were shown as a reference (left panel).
    Sp1 D4c3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc rabbit monoclonal ab against sp1
    ZEB1 activates VEGFA transcription by recruiting <t>SP1</t> to the endogenous VEGFA promoter. (A) Sequential deletion and mutation of SP1 elements on the human VEGFA promoter were fused to the luciferase reporter. (B) MDA-MB-231 cells were co-transfected with the ZEB1 expression plasmid (1 μg/well) and different wild-type VEGFA promoter luciferase reporter constructs (1 μg/well). Extract luciferase activities were determined 48 h after transfection using a Betascope analyzer. Luciferase values were normalized to Renilla activities. * P
    Rabbit Monoclonal Ab Against Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Schematic model of BMX regulates VEGFR2 transcription by interaction with Sp1. The SH2/SH3 domain of BMX confers its nuclear localization in ECs. Nuclear BMX in an active form interacts with (and possibly phosphorylates) Sp1 to facilitate the recruitment of Sp1 to the VEGFR2 promoter and its transcription in ECs

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression, et al. Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression

    doi: 10.1111/jcmm.14663

    Figure Lengend Snippet: Schematic model of BMX regulates VEGFR2 transcription by interaction with Sp1. The SH2/SH3 domain of BMX confers its nuclear localization in ECs. Nuclear BMX in an active form interacts with (and possibly phosphorylates) Sp1 to facilitate the recruitment of Sp1 to the VEGFR2 promoter and its transcription in ECs

    Article Snippet: The antibodies included anti‐Sp1 (#9389; Cell Signaling Technology), a positive control (Anti‐RNA Polymerase II) and a negative control (normal IgG) that were employed for each immunoprecipitation.

    Techniques:

    Active BMX interacts with Sp1 in the nucleus and facilitates Sp1 binding to the Vegfr2 promoter. A, Schematic diagram for the Sp1 binding sites located on the Vegfr2 promoter. −123 to −46 are positions related to the transcription start site (TSS; +1). B, BMX promotes Sp1 binding to the Vegfr2 promoter. HDLECs were transfected with human BMX siRNA or control siRNA (20 nmol/L) for 48 h. ChIP assay was then performed with Sp1 antibody. An Sp1 binding region of the Vegfr2 promoter was used as a primer for quantitative PCR. C, HUVECs were cotransfected with a Vegfr2 reporter (−123 to +1), Renilla luciferase plasmid and either vector control (Vector), BMX‐WT or kinase‐dead K445R‐BMX alone or together with Sp1 for 48 h. The dual‐luciferase assay was then performed. The firefly luciferase readout was normalized to that of Renilla luciferase. The data are means ± SEM from three independent experiments. **, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression, et al. Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression

    doi: 10.1111/jcmm.14663

    Figure Lengend Snippet: Active BMX interacts with Sp1 in the nucleus and facilitates Sp1 binding to the Vegfr2 promoter. A, Schematic diagram for the Sp1 binding sites located on the Vegfr2 promoter. −123 to −46 are positions related to the transcription start site (TSS; +1). B, BMX promotes Sp1 binding to the Vegfr2 promoter. HDLECs were transfected with human BMX siRNA or control siRNA (20 nmol/L) for 48 h. ChIP assay was then performed with Sp1 antibody. An Sp1 binding region of the Vegfr2 promoter was used as a primer for quantitative PCR. C, HUVECs were cotransfected with a Vegfr2 reporter (−123 to +1), Renilla luciferase plasmid and either vector control (Vector), BMX‐WT or kinase‐dead K445R‐BMX alone or together with Sp1 for 48 h. The dual‐luciferase assay was then performed. The firefly luciferase readout was normalized to that of Renilla luciferase. The data are means ± SEM from three independent experiments. **, P

    Article Snippet: The antibodies included anti‐Sp1 (#9389; Cell Signaling Technology), a positive control (Anti‐RNA Polymerase II) and a negative control (normal IgG) that were employed for each immunoprecipitation.

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

    Working hypothesis whereby drug‐eluting stent targeting Sp‐1 (specificity protein 1) inhibited neointimal formation via regulating YAP (Yes‐associated protein)‐mediated smooth muscle cell (SMC) phenotypic switch. A , An accumulation of synthetic SMCs in the stented artery contributed to in‐stent restenosis. Sp‐1 inhibitor eluted from the stent regulates SMC from synthetic to contractile state. B , Mithramycin A downregulates YAP expression through inhibiting transcription factor Sp‐1. Reduced YAP expression facilitates MyocD/SRF interaction and thereby maintains contractile gene expression (SMMHC, SM22α). PDGF‐BB indicates platelet‐derived growth factor BB; SMMHC, SM myosin heavy chain.

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Working hypothesis whereby drug‐eluting stent targeting Sp‐1 (specificity protein 1) inhibited neointimal formation via regulating YAP (Yes‐associated protein)‐mediated smooth muscle cell (SMC) phenotypic switch. A , An accumulation of synthetic SMCs in the stented artery contributed to in‐stent restenosis. Sp‐1 inhibitor eluted from the stent regulates SMC from synthetic to contractile state. B , Mithramycin A downregulates YAP expression through inhibiting transcription factor Sp‐1. Reduced YAP expression facilitates MyocD/SRF interaction and thereby maintains contractile gene expression (SMMHC, SM22α). PDGF‐BB indicates platelet‐derived growth factor BB; SMMHC, SM myosin heavy chain.

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Expressing, Derivative Assay

    Sp‐1 (specificity protein 1) promoted smooth muscle cell (SMC) proliferation and migration. A , SMCs were transfected with Sp‐1 plasmid which expressed a high level of Sp‐1. Proliferation of SMCs was measured in a variety of cell culture media using 4‐5‐dimethyl‐2‐thiazolyl‐2‐5‐diphenyl‐2H‐tetrazolium bromide assay as indicated. * P

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Sp‐1 (specificity protein 1) promoted smooth muscle cell (SMC) proliferation and migration. A , SMCs were transfected with Sp‐1 plasmid which expressed a high level of Sp‐1. Proliferation of SMCs was measured in a variety of cell culture media using 4‐5‐dimethyl‐2‐thiazolyl‐2‐5‐diphenyl‐2H‐tetrazolium bromide assay as indicated. * P

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Migration, Transfection, Plasmid Preparation, Cell Culture

    Sp‐1 (specificity protein 1) increased YAP (Yes‐associated protein) promoter activity in the process of SMC phenotypic modulation. A , Schematic showing the series of YAP promoter‐reporter plasmids used. B , Smooth muscle cells (SMCs) were transfected with the 2200‐, 1150‐, 450‐, or 50‐bp YAP promoter‐luciferase (Luc) plasmid and further transfected with vector or Sp‐1 plasmid before determining relative changes in luciferase expression. Data indicated that Sp‐1 overexpression‐mediated increases in YAP promoter activity required sequences between −450 and −50 bp. * P

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Sp‐1 (specificity protein 1) increased YAP (Yes‐associated protein) promoter activity in the process of SMC phenotypic modulation. A , Schematic showing the series of YAP promoter‐reporter plasmids used. B , Smooth muscle cells (SMCs) were transfected with the 2200‐, 1150‐, 450‐, or 50‐bp YAP promoter‐luciferase (Luc) plasmid and further transfected with vector or Sp‐1 plasmid before determining relative changes in luciferase expression. Data indicated that Sp‐1 overexpression‐mediated increases in YAP promoter activity required sequences between −450 and −50 bp. * P

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Over Expression

    Sp‐1 (specificity protein 1) was upregulated in smooth muscle cell (SMC) phenotypic modulation both in vitro and in vivo. A , Platelet‐derived growth factor‐BB (PDGF‐BB) induced Sp‐1 expression and attenuated contractile protein expression in SMCs. SMCs were starved in DMEM without fetal bovine serum (FBS) for 24 h before being treated with vehicle or PDGF‐BB (1, 10, and 20 ng/mL). Western blotting was performed to examine the expression of Sp‐1, SM22α, SM myosin heavy chain (SMMHC), calponin, and proliferation marker cyclin D. B , Quantitative analysis of mRNA expression. * P

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Sp‐1 (specificity protein 1) was upregulated in smooth muscle cell (SMC) phenotypic modulation both in vitro and in vivo. A , Platelet‐derived growth factor‐BB (PDGF‐BB) induced Sp‐1 expression and attenuated contractile protein expression in SMCs. SMCs were starved in DMEM without fetal bovine serum (FBS) for 24 h before being treated with vehicle or PDGF‐BB (1, 10, and 20 ng/mL). Western blotting was performed to examine the expression of Sp‐1, SM22α, SM myosin heavy chain (SMMHC), calponin, and proliferation marker cyclin D. B , Quantitative analysis of mRNA expression. * P

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: In Vitro, In Vivo, Derivative Assay, Expressing, Western Blot, Marker

    Fabrication of Sp‐1 (specificity protein 1) inhibitor Mithramycin A (MTA)‐eluting stent and stent implantation. A , Scanning electron microscope (SEM) images of BMS and MTA‐eluting stent (MES). # Indicates the coating surface of the stents, which was smooth and uniform. B , Cumulative release profile of Mithramycin A from MES. C , Stents were successfully implanted in the rabbit carotid model. # Indicates the location of the stent. D , Postoperative radiograph demonstrates no stent migration and fracture in the following time. Arrow in the figure indicates the location of the stent.

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Fabrication of Sp‐1 (specificity protein 1) inhibitor Mithramycin A (MTA)‐eluting stent and stent implantation. A , Scanning electron microscope (SEM) images of BMS and MTA‐eluting stent (MES). # Indicates the coating surface of the stents, which was smooth and uniform. B , Cumulative release profile of Mithramycin A from MES. C , Stents were successfully implanted in the rabbit carotid model. # Indicates the location of the stent. D , Postoperative radiograph demonstrates no stent migration and fracture in the following time. Arrow in the figure indicates the location of the stent.

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Microscopy, Migration

    Sp‐1 (specificity protein 1) inhibitor‐eluting stent attenuated restenosis through targeting smooth muscle cell phenotypic modulation. A , Representative images of hematoxylin and eosin staining of cross sections 6 mo after implantation. Mithramycin A‐eluting stent (MES) had significantly reduced neointimal formation compared with bare metal stent (BMS). B , Statistical analysis of average intimal thickness and areas of neointima of the stents. * P

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: Sp‐1 (specificity protein 1) inhibitor‐eluting stent attenuated restenosis through targeting smooth muscle cell phenotypic modulation. A , Representative images of hematoxylin and eosin staining of cross sections 6 mo after implantation. Mithramycin A‐eluting stent (MES) had significantly reduced neointimal formation compared with bare metal stent (BMS). B , Statistical analysis of average intimal thickness and areas of neointima of the stents. * P

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Staining

    YAP (Yes‐associated protein) was involved in Sp‐1 (specificity protein 1)‐mediated SMC phenotypic modulation. A and B , Platelet‐derived growth factor‐BB (PDGF‐BB, 20 ng/mL for 24 h) induced YAP protein ( A ) and mRNA ( B ) expression in parallel with Sp‐1 expression. Additional siSp‐1 transfected or Mithramycin A (MTA) treatment could reverse this process. * P

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

    Article Title: Drug‐Eluting Stent Targeting Sp‐1‐Attenuated Restenosis by Engaging YAP‐Mediated Vascular Smooth Muscle Cell Phenotypic Modulation

    doi: 10.1161/JAHA.119.014103

    Figure Lengend Snippet: YAP (Yes‐associated protein) was involved in Sp‐1 (specificity protein 1)‐mediated SMC phenotypic modulation. A and B , Platelet‐derived growth factor‐BB (PDGF‐BB, 20 ng/mL for 24 h) induced YAP protein ( A ) and mRNA ( B ) expression in parallel with Sp‐1 expression. Additional siSp‐1 transfected or Mithramycin A (MTA) treatment could reverse this process. * P

    Article Snippet: Antibodies used were as follows: anti‐Sp‐1 (CST; catalog no. #9389), anti‐YAP (CST; catalog no. #14074), anti‐SM22α (Abcam; catalog no. ab10135), anti‐calponin (Abcam; catalog no. ab46794), anti‐SMMHC (Abcam; catalog no. ab52319), anti‐Cyclin D (CST; catalog no. #2978), anti‐GAPDH (CST; catalog no. #5174).

    Techniques: Derivative Assay, Expressing, Transfection

    Sp1 down-regulation results in decreased expression of prosurvival genes. Mithramycin ( A ) and siRNA Sp1 ( siSp1 ; B ) down-regulation of HSP70, HSF1, and HSP27. Both HSF1 ( C ) and NF-κB ( D ) activities were reduced after Sp1 inhibition. Sp1 ( E and

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Sp1 down-regulation results in decreased expression of prosurvival genes. Mithramycin ( A ) and siRNA Sp1 ( siSp1 ; B ) down-regulation of HSP70, HSF1, and HSP27. Both HSF1 ( C ) and NF-κB ( D ) activities were reduced after Sp1 inhibition. Sp1 ( E and

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: Expressing, Inhibition

    Triptolide inhibits O -GlcNAc modification of Sp1. A , immunoprecipitation ( Ip ) showing that Sp1 was glycosylated with O -GlcNAc modification and decreased after treatment with triptolide. S , supernatant; Ip , immunoprecipitated pellet. B , inhibition of

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Triptolide inhibits O -GlcNAc modification of Sp1. A , immunoprecipitation ( Ip ) showing that Sp1 was glycosylated with O -GlcNAc modification and decreased after treatment with triptolide. S , supernatant; Ip , immunoprecipitated pellet. B , inhibition of

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: Modification, Immunoprecipitation, Inhibition

    Sp1 is overexpressed in pancreatic cancer. A , Sp1 RNA expression in different pancreatic cancer cell lines compared with normal pancreatic ductal cells. B , Sp1 protein expression in pancreatic cancer cell lines compared with human pancreatic ductal cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Sp1 is overexpressed in pancreatic cancer. A , Sp1 RNA expression in different pancreatic cancer cell lines compared with normal pancreatic ductal cells. B , Sp1 protein expression in pancreatic cancer cell lines compared with human pancreatic ductal cells.

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: RNA Expression, Expressing

    Sp1 overexpression rescues pancreatic cells from triptolide-induced death. A , clones Sp1-NLS1 and Sp1-NLS2 (expressing the NLS), Sp1 overexpression (without NLS), OGT overexpression, and stimulation of cells with 5 m m glucosamine rescued pancreatic cancer

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Sp1 overexpression rescues pancreatic cells from triptolide-induced death. A , clones Sp1-NLS1 and Sp1-NLS2 (expressing the NLS), Sp1 overexpression (without NLS), OGT overexpression, and stimulation of cells with 5 m m glucosamine rescued pancreatic cancer

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: Over Expression, Clone Assay, Expressing

    Sp1 inhibition down-regulates NF-κB and HSF1 promoter binding activity. A , inhibition of Sp1 by siRNA ( si Sp1 ) or mithramycin resulted in down-regulation of NF-κB activity. BAY11-7085, a NF-κB pathway inhibitor, was used as a control.

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Sp1 inhibition down-regulates NF-κB and HSF1 promoter binding activity. A , inhibition of Sp1 by siRNA ( si Sp1 ) or mithramycin resulted in down-regulation of NF-κB activity. BAY11-7085, a NF-κB pathway inhibitor, was used as a control.

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: Inhibition, Binding Assay, Activity Assay

    Sp1 down-regulation results in loss of viability in cancer cells. Mithramycin treatment ( A ) or Sp1 inhibition by siRNA ( B ) resulted in MIA-PaCa2 and S2-013 cell death and decreased proliferation in MIA-PaCa2 ( C ) and S2-013 ( D ) cells. E , tumor regression

    Journal: The Journal of Biological Chemistry

    Article Title: Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1 *

    doi: 10.1074/jbc.M113.500983

    Figure Lengend Snippet: Sp1 down-regulation results in loss of viability in cancer cells. Mithramycin treatment ( A ) or Sp1 inhibition by siRNA ( B ) resulted in MIA-PaCa2 and S2-013 cell death and decreased proliferation in MIA-PaCa2 ( C ) and S2-013 ( D ) cells. E , tumor regression

    Article Snippet: The immunoprecipitated samples were immunoblotted with anti- O -GlcNAc and anti-Sp1 antibodies.

    Techniques: Inhibition

    ESR1-mediated stimulation of Slc2a4 gene transcription involves a SP1's cooperative mechanism. A : ESR-binding and SP1-binding consensus sequences 20 , 35 . B : -239/-40 segment of Slc2a4 promoter depicting: the -149/-125 sequence used for EMSA analysis (in the box), containing the SP1-binding site (shaded); and 2 large sequences homologous to the complete (palindromic) ESR-binding site, 3 short sequences homologous to the first half-site of the ESR-binding site and 1 short sequence homologous to the second half-site of the ESR-binding site (underlined). C : EMSA analysis of SP1 binding into the -149/-125 segment of Slc2a4 gene promoter. D : SP1 binding activity into Slc2a4 promoter measured in 3T3-L1 cells 24-hour treated in culture medium alone (C) or supplemented with estradiol (E2), ESR1 selective agonist (PPT) or both (PPT+E2). At the top, representative experiment shows blots of the SP1/DNA complexes, in the same sequence of the graph bars. Data are means ± SEM of 5 different samples, compared by one-way-ANOVA, followed by Tukey's post-test, after to confirm the normality of the data distribution by the Shapiro-Wilk test. **P

    Journal: International Journal of Medical Sciences

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism

    doi: 10.7150/ijms.26774

    Figure Lengend Snippet: ESR1-mediated stimulation of Slc2a4 gene transcription involves a SP1's cooperative mechanism. A : ESR-binding and SP1-binding consensus sequences 20 , 35 . B : -239/-40 segment of Slc2a4 promoter depicting: the -149/-125 sequence used for EMSA analysis (in the box), containing the SP1-binding site (shaded); and 2 large sequences homologous to the complete (palindromic) ESR-binding site, 3 short sequences homologous to the first half-site of the ESR-binding site and 1 short sequence homologous to the second half-site of the ESR-binding site (underlined). C : EMSA analysis of SP1 binding into the -149/-125 segment of Slc2a4 gene promoter. D : SP1 binding activity into Slc2a4 promoter measured in 3T3-L1 cells 24-hour treated in culture medium alone (C) or supplemented with estradiol (E2), ESR1 selective agonist (PPT) or both (PPT+E2). At the top, representative experiment shows blots of the SP1/DNA complexes, in the same sequence of the graph bars. Data are means ± SEM of 5 different samples, compared by one-way-ANOVA, followed by Tukey's post-test, after to confirm the normality of the data distribution by the Shapiro-Wilk test. **P

    Article Snippet: Primary anti-SP1 (# 5931, Cell Signaling), anti-ESR1 (#06-935, Millipore) and anti-ESR2 (#sc-6821, Santa Cruz Biotechnology Inc.) antibodies were used, followed by the appropriate secondary antibody and final chemiluminescence analysis.

    Techniques: Electron Paramagnetic Resonance, Binding Assay, Sequencing, Activity Assay

    ESR1 activity increases Slc2a4/GLUT4 expression and nuclear content of SP1 in adipocytes Adipocytes (3T3-L1) were treated (24 hours) with no stimulus (C), with estradiol (E2), ESR1 agonist alone (PPT) or with E2 (PPT+E2), and ESR2 agonist alone (DPN) or with E2 (DPN+E2). Slc2a4 mRNA (A), total cellular GLUT4 protein (B), nuclear ESR1 (C), nuclear ESR2 (D), Sp1 mRNA (E) and nuclear SP1 (F) contents were measured. For each protein analyzed, a representative immunoblot and respective Ponceau stained membrane are shown; lanes are in the same sequence of the graph bars. Data are means ± SEM of 5 different samples, compared by one-way-ANOVA, followed by Tukey's post-test, after to confirm the normality of the data distribution by the Shapiro-Wilk test. *P

    Journal: International Journal of Medical Sciences

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism

    doi: 10.7150/ijms.26774

    Figure Lengend Snippet: ESR1 activity increases Slc2a4/GLUT4 expression and nuclear content of SP1 in adipocytes Adipocytes (3T3-L1) were treated (24 hours) with no stimulus (C), with estradiol (E2), ESR1 agonist alone (PPT) or with E2 (PPT+E2), and ESR2 agonist alone (DPN) or with E2 (DPN+E2). Slc2a4 mRNA (A), total cellular GLUT4 protein (B), nuclear ESR1 (C), nuclear ESR2 (D), Sp1 mRNA (E) and nuclear SP1 (F) contents were measured. For each protein analyzed, a representative immunoblot and respective Ponceau stained membrane are shown; lanes are in the same sequence of the graph bars. Data are means ± SEM of 5 different samples, compared by one-way-ANOVA, followed by Tukey's post-test, after to confirm the normality of the data distribution by the Shapiro-Wilk test. *P

    Article Snippet: Primary anti-SP1 (# 5931, Cell Signaling), anti-ESR1 (#06-935, Millipore) and anti-ESR2 (#sc-6821, Santa Cruz Biotechnology Inc.) antibodies were used, followed by the appropriate secondary antibody and final chemiluminescence analysis.

    Techniques: Activity Assay, Expressing, Staining, Sequencing

    FAS rs2234767 A and rs1800682 G alleles affect coupled SP1 and STAT1 recruitment to chromatin. ( A ) Chromatin immunoprecipitation (ChIP) of the FAS promoter with three different genotypes (rs2234767 GG/rs1800682 AA, rs2234767 GA/rs1800682 AG and rs2234767 AA/rs1800682 GG) using antibody for SP1 and STAT1(single pool generated from triplicate biological samples/manipulation; triplicate measurements/pool; mean ± SE). ( B ) Sequential ChIP of the FAS promoter containing SP1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). ( C ) Sequential ChIP of the FAS promoter containing STAT1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). For all combined genotypes in the figure, left genotype arises from the rs2234767 and right from the rs1800682 polymorphism.

    Journal: Scientific Reports

    Article Title: FAS rs2234767 and rs1800682 polymorphisms jointly contributed to risk of colorectal cancer by affecting SP1/STAT1 complex recruitment to chromatin

    doi: 10.1038/srep19229

    Figure Lengend Snippet: FAS rs2234767 A and rs1800682 G alleles affect coupled SP1 and STAT1 recruitment to chromatin. ( A ) Chromatin immunoprecipitation (ChIP) of the FAS promoter with three different genotypes (rs2234767 GG/rs1800682 AA, rs2234767 GA/rs1800682 AG and rs2234767 AA/rs1800682 GG) using antibody for SP1 and STAT1(single pool generated from triplicate biological samples/manipulation; triplicate measurements/pool; mean ± SE). ( B ) Sequential ChIP of the FAS promoter containing SP1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). ( C ) Sequential ChIP of the FAS promoter containing STAT1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). For all combined genotypes in the figure, left genotype arises from the rs2234767 and right from the rs1800682 polymorphism.

    Article Snippet: The antisera for ChIP reaction was normal mouse IgG (Cat. No. 2027, Santa Cruz Biotechnology, Inc.), normal rabbit IgG (Cat. No. NI01, EMD Chemicals, Inc., Gibbstown, NJ), anti-human SP1 (Cat. No. 9389, Cell Signaling Technology), and anti-human STAT1 (Cat. No. 9172, Cell Signaling Technology).

    Techniques: Chromatin Immunoprecipitation, Generated, Immunoprecipitation

    Sp1 regulated the transcription of the XRCC1 by recruiting Krox-20 to the rs3231245 mutation region (A, B, D, E, G and H) Binding affinity of Sp1 and Krox-20 to chromatin. (C, F and I) Confirmation of protein expression of Sp1 and Krox-20 in siRNA-transfected cells by Western blot analysis.

    Journal: Oncotarget

    Article Title: XRCC1 mediated the development of cervival cancer through a novel Sp1/Krox-20 swich

    doi: 10.18632/oncotarget.21040

    Figure Lengend Snippet: Sp1 regulated the transcription of the XRCC1 by recruiting Krox-20 to the rs3231245 mutation region (A, B, D, E, G and H) Binding affinity of Sp1 and Krox-20 to chromatin. (C, F and I) Confirmation of protein expression of Sp1 and Krox-20 in siRNA-transfected cells by Western blot analysis.

    Article Snippet: Three primary antibodies were employed in the experiments: human Sp1 (1:1000 dilution; CST, USA), Krox-20(1:1000 dilution; abcam, USA), and α-tubulin (1:1000 dilution; Sigma, USA).

    Techniques: Mutagenesis, Binding Assay, Expressing, Transfection, Western Blot

    Schematic model of the regulations among rs3213245 and XRCC1 involved in cervical cancer development The transcription factor Krox-20 was recruited to the Sp1 binding motif. The rs3213245 C > T polymorphism may change the binding affinity of the transcription factor Sp1-Krox-20 complex to the mutation region, thereby regulating the expression of the XRCC1 gene and ultimately leads to cervical cancer development.

    Journal: Oncotarget

    Article Title: XRCC1 mediated the development of cervival cancer through a novel Sp1/Krox-20 swich

    doi: 10.18632/oncotarget.21040

    Figure Lengend Snippet: Schematic model of the regulations among rs3213245 and XRCC1 involved in cervical cancer development The transcription factor Krox-20 was recruited to the Sp1 binding motif. The rs3213245 C > T polymorphism may change the binding affinity of the transcription factor Sp1-Krox-20 complex to the mutation region, thereby regulating the expression of the XRCC1 gene and ultimately leads to cervical cancer development.

    Article Snippet: Three primary antibodies were employed in the experiments: human Sp1 (1:1000 dilution; CST, USA), Krox-20(1:1000 dilution; abcam, USA), and α-tubulin (1:1000 dilution; Sigma, USA).

    Techniques: Binding Assay, Mutagenesis, Expressing

    SP1 was a directly targeted gene of miR-149 in vitro . (A) Bioinformatics software assessed that SP1 mRNA contained a miR-149 seed match at position 3809–3815 of the SP1 3′-UTR. (B) miR-149 significantly inhibited the SP1 Wt but not the

    Journal: Oncology Letters

    Article Title: MicroRNA-149 targets specificity protein 1 to suppress human tongue squamous cell carcinoma cell proliferation and motility

    doi: 10.3892/ol.2016.5527

    Figure Lengend Snippet: SP1 was a directly targeted gene of miR-149 in vitro . (A) Bioinformatics software assessed that SP1 mRNA contained a miR-149 seed match at position 3809–3815 of the SP1 3′-UTR. (B) miR-149 significantly inhibited the SP1 Wt but not the

    Article Snippet: The membranes were blocked with 5% skimmed milk at room temperature for 2 h and incubated with rabbit anti-human SP1 antibody (dilution, 1: 1,000; cat. no., 5931; Cell Signaling Technology, Inc., Danvers, MA, USA) according to the protocol of the manufacturer at 4°C overnight.

    Techniques: In Vitro, Software

    p16 modulates the expression of different Sp1 targets. A and C , total RNA was prepared from the indicated cells, and quantitative RT-PCR was performed using specific primers for the indicated genes. B , EMSA was performed using nuclear extracts from the

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: p16 modulates the expression of different Sp1 targets. A and C , total RNA was prepared from the indicated cells, and quantitative RT-PCR was performed using specific primers for the indicated genes. B , EMSA was performed using nuclear extracts from the

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Expressing, Quantitative RT-PCR

    p16 positively regulates the expression of miR-141 and miR-146b-5p through the Sp1 transcription factor. A , nucleotide sequences of the miR-141 and miR-146b-5p promoters, −39 to +9 nucleotides and −93 to −26 nucleotides, respectively,

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: p16 positively regulates the expression of miR-141 and miR-146b-5p through the Sp1 transcription factor. A , nucleotide sequences of the miR-141 and miR-146b-5p promoters, −39 to +9 nucleotides and −93 to −26 nucleotides, respectively,

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Expressing

    p16, Sp1, and CDK4 proteins form a heterocomplex that binds the miR-141 and miR-146b-5p promoters. A , whole cell extracts were prepared from HFSN1 cells and immunoprecipitated ( IP ) with anti-p16, anti-Sp1, or anti-CDK4 antibodies (mouse IgG was used as

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: p16, Sp1, and CDK4 proteins form a heterocomplex that binds the miR-141 and miR-146b-5p promoters. A , whole cell extracts were prepared from HFSN1 cells and immunoprecipitated ( IP ) with anti-p16, anti-Sp1, or anti-CDK4 antibodies (mouse IgG was used as

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Immunoprecipitation

    p16 interacts with Sp1 through the fourth ankyrin repeat. A , p16-negative U2OS cells were transfected with the indicated constructs, and then whole cell extracts were prepared and used for immunoprecipitation ( IP ) with anti-p16 antibody (mouse IgG was

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: p16 interacts with Sp1 through the fourth ankyrin repeat. A , p16-negative U2OS cells were transfected with the indicated constructs, and then whole cell extracts were prepared and used for immunoprecipitation ( IP ) with anti-p16 antibody (mouse IgG was

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Transfection, Construct, Immunoprecipitation

    Sp1 binds the miR-141 and miR-146b-5p promoters and activates their transcription in a p16-dependent manner. A , ChIP assay. Chromatin was purified from HFSN1C and HFSN1p16sh, and then immunoprecipitated ( IP ) using anti-Sp1 antibody. miR-141 and miR-146b-5p

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: Sp1 binds the miR-141 and miR-146b-5p promoters and activates their transcription in a p16-dependent manner. A , ChIP assay. Chromatin was purified from HFSN1C and HFSN1p16sh, and then immunoprecipitated ( IP ) using anti-Sp1 antibody. miR-141 and miR-146b-5p

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Chromatin Immunoprecipitation, Purification, Immunoprecipitation

    p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p and their role in apoptosis following UV damage. A , cells were either mock-treated or challenged with UV light (10 Jm −2 ) and then re-incubated for the indicated periods of time. Total

    Journal: The Journal of Biological Chemistry

    Article Title: The Cyclin-dependent Kinase Inhibitor p16INK4a Physically Interacts with Transcription Factor Sp1 and Cyclin-dependent Kinase 4 to Transactivate MicroRNA-141 and MicroRNA-146b-5p Spontaneously and in Response to Ultraviolet Light-induced DNA Damage *

    doi: 10.1074/jbc.M113.512640

    Figure Lengend Snippet: p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p and their role in apoptosis following UV damage. A , cells were either mock-treated or challenged with UV light (10 Jm −2 ) and then re-incubated for the indicated periods of time. Total

    Article Snippet: ChIP experiments were performed using antibodies against Sp1 (Cell Signaling Technology), p16 (BD Biosciences), and CDK4 (C-22).

    Techniques: Incubation

    Nrf2 enhances the mRNA transcription of PDGFA by binding to PDGFA promoter via Sp1 ( A ) Ablation of Nrf2 downregulated PDGFA mRNA expression. MHCC-97H and Hep3B cells were infected with indicated lentivirus and the mRNA levels of PDGFA in indicated group were measured by qRT-PCR. ( B ) Ectopic expression of Nrf2 increased PDGFA mRNA expression. The mRNA levels of PDGFA in indicated group were measured by qRT-PCR. Means ± SD from three independent experiments are presented as relative ratio to the control whose value was taken as 1.0. ( C ) Nrf2 activated PDGFA promoter. MHCC-97H cells were co-transfected with indicated plasmids for 48 h. Means ± SD of normalized luciferase activity from three independent experiments are presented. ( D ) Nrf2 dose-dependently activated PDGFA promoter. HEK-293T cells cotransfected with PGL3-PDGFA, pRL-SV40, and various amounts (0, 100, 200, 400, 800 ng/well) of pcDNA-Nrf2 or pcDNA3.0 vectors. Means ± SD of normalized luciferase activity from three independent experiments are presented. ( E ) Nrf2 could bind to PDGFA promoter. ChIP assays were performed by immunoprecipitation chromatin fragments from MHCC-97H cells using anti-Nrf2 mAb or IgG control. 5% cell lysate was used as input. ( F ) Characterization of the region −223/+20 in PDGFA promoter revealed four binding sites for Sp1 (marked in red box). ( G ) Nrf2 interacts with Sp1 in the nucleus. Nucleoproteins were then extracted and subjected to Co-IP assay analysis. In this way we further revealed the interactions between Nrf2 and Sp1. 5% cell lysate was used as input. IB, immunoblot. IP, immunoprecipitation. ( H ) PDGFA luciferase activities were detected under the NC and Nrf2 overexpression group, respectively. Cells were transfected with no siRNAs (control), or Sp1 siRNA (siSp1). Means ± s.d. of normalized luciferase activity from three independent experiments are presented. ( I ) The upregulation of PDGFA by Nrf2 was dependent on Sp1. Cells that were infected with the indicated lentivirus were transfected with the indicated siSp1. The expression of PDGFA was measured by qRT-PCR and western blotting. ( J ) Sp1 is essential for Nrf2 binding to PDGFA promoter. ChIP assays were performed by immunoprecipitation chromatin fragments from MHCC-97H cells with Mock group or Sp1 depletion group using anti-Nrf2 mAb or IgG control. 5% cell lysate was used as input. ( K ) Nrf2 is required for Sp1 enrichment on PDGFA promoter. ChIP assays were performed with antibody against Sp1 or control IgG in MHCC-97H cells expressed high Nrf2 level or low Nrf2 level. Left, Nrf2 expression promotes Sp1 association with PDGFA promoter. Right, Nrf2 ablation attenuates Sp1 interaction with PDGFA promoter. * P

    Journal: Oncotarget

    Article Title: Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression

    doi: 10.18632/oncotarget.11700

    Figure Lengend Snippet: Nrf2 enhances the mRNA transcription of PDGFA by binding to PDGFA promoter via Sp1 ( A ) Ablation of Nrf2 downregulated PDGFA mRNA expression. MHCC-97H and Hep3B cells were infected with indicated lentivirus and the mRNA levels of PDGFA in indicated group were measured by qRT-PCR. ( B ) Ectopic expression of Nrf2 increased PDGFA mRNA expression. The mRNA levels of PDGFA in indicated group were measured by qRT-PCR. Means ± SD from three independent experiments are presented as relative ratio to the control whose value was taken as 1.0. ( C ) Nrf2 activated PDGFA promoter. MHCC-97H cells were co-transfected with indicated plasmids for 48 h. Means ± SD of normalized luciferase activity from three independent experiments are presented. ( D ) Nrf2 dose-dependently activated PDGFA promoter. HEK-293T cells cotransfected with PGL3-PDGFA, pRL-SV40, and various amounts (0, 100, 200, 400, 800 ng/well) of pcDNA-Nrf2 or pcDNA3.0 vectors. Means ± SD of normalized luciferase activity from three independent experiments are presented. ( E ) Nrf2 could bind to PDGFA promoter. ChIP assays were performed by immunoprecipitation chromatin fragments from MHCC-97H cells using anti-Nrf2 mAb or IgG control. 5% cell lysate was used as input. ( F ) Characterization of the region −223/+20 in PDGFA promoter revealed four binding sites for Sp1 (marked in red box). ( G ) Nrf2 interacts with Sp1 in the nucleus. Nucleoproteins were then extracted and subjected to Co-IP assay analysis. In this way we further revealed the interactions between Nrf2 and Sp1. 5% cell lysate was used as input. IB, immunoblot. IP, immunoprecipitation. ( H ) PDGFA luciferase activities were detected under the NC and Nrf2 overexpression group, respectively. Cells were transfected with no siRNAs (control), or Sp1 siRNA (siSp1). Means ± s.d. of normalized luciferase activity from three independent experiments are presented. ( I ) The upregulation of PDGFA by Nrf2 was dependent on Sp1. Cells that were infected with the indicated lentivirus were transfected with the indicated siSp1. The expression of PDGFA was measured by qRT-PCR and western blotting. ( J ) Sp1 is essential for Nrf2 binding to PDGFA promoter. ChIP assays were performed by immunoprecipitation chromatin fragments from MHCC-97H cells with Mock group or Sp1 depletion group using anti-Nrf2 mAb or IgG control. 5% cell lysate was used as input. ( K ) Nrf2 is required for Sp1 enrichment on PDGFA promoter. ChIP assays were performed with antibody against Sp1 or control IgG in MHCC-97H cells expressed high Nrf2 level or low Nrf2 level. Left, Nrf2 expression promotes Sp1 association with PDGFA promoter. Right, Nrf2 ablation attenuates Sp1 interaction with PDGFA promoter. * P

    Article Snippet: The cross-linked protein- DNA complexes were immunoprecipitated using an anti-Nrf2 antibody (Abcam, Cambridge, UK) and Sp1 antibody (#5931, Cell Signaling Technology).

    Techniques: Binding Assay, Expressing, Infection, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Western Blot

    Insulin, high glucose and FFA increase Inpp5f expression by different transcription factors. ( A ) Comparison of the gene sequences of Inpp5f among different species using web-based ECR (see article in ref. 35 ). ( B ) The 500 bp promoter region and the first exon of Inpp5f in different species were screened for the putative Sp1 and NF-κB binding sites by JASPAR web (see article in ref. 36 ). ( C ) Serum starved H9C2 cells were treated with Insulin and expression of Sp1 was tested by Western blot. ( D ) H9C2 cells were treated with HG or FFA for 24 hrs, p65 nuclear translocation was analyzed by western blot in cytoplasmic and nuclear extract respectively. ( E,F ) H9C2 cells were treated with insulin, Sp1 inhibitor or the combination. Inpp5f expression at mRNA ( E ) and protein levels ( F ) was examined by qPCR and Western Blot. *P

    Journal: Scientific Reports

    Article Title: Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart

    doi: 10.1038/srep22068

    Figure Lengend Snippet: Insulin, high glucose and FFA increase Inpp5f expression by different transcription factors. ( A ) Comparison of the gene sequences of Inpp5f among different species using web-based ECR (see article in ref. 35 ). ( B ) The 500 bp promoter region and the first exon of Inpp5f in different species were screened for the putative Sp1 and NF-κB binding sites by JASPAR web (see article in ref. 36 ). ( C ) Serum starved H9C2 cells were treated with Insulin and expression of Sp1 was tested by Western blot. ( D ) H9C2 cells were treated with HG or FFA for 24 hrs, p65 nuclear translocation was analyzed by western blot in cytoplasmic and nuclear extract respectively. ( E,F ) H9C2 cells were treated with insulin, Sp1 inhibitor or the combination. Inpp5f expression at mRNA ( E ) and protein levels ( F ) was examined by qPCR and Western Blot. *P

    Article Snippet: After blocking with 5% non-fat milk, the membranes were probed overnight at 4 °C with Inpp5f (1:500, cat# SAB2700848, Sigma, USA), p-Akt Ser473 (1:1000, cat# 9271, Cell signaling, USA), total Akt (1:1000, cat# 9272, Cell signaling, USA), p65 (1:1000, ab32536, Abcam, USA), Sp1 (1:1000, cat# 5931, Cell signaling, USA), β-actin (1:1000, cat# AB10024, Sangon, China), Lamin B (1:500, cat# BA1228, Boster, China) antibodies.

    Techniques: Expressing, Binding Assay, Western Blot, Translocation Assay, Real-time Polymerase Chain Reaction

    Sp1 and NF-κB transcriptionally activate Inpp5f expression. ( A ) H9C2 cells were co-transfected with mouse Inpp5f promoter reporter (pGL3-Inp) and pRL-TK and additionally treated with ctrl, insulin with/or Sp1 inhibitor. *P

    Journal: Scientific Reports

    Article Title: Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart

    doi: 10.1038/srep22068

    Figure Lengend Snippet: Sp1 and NF-κB transcriptionally activate Inpp5f expression. ( A ) H9C2 cells were co-transfected with mouse Inpp5f promoter reporter (pGL3-Inp) and pRL-TK and additionally treated with ctrl, insulin with/or Sp1 inhibitor. *P

    Article Snippet: After blocking with 5% non-fat milk, the membranes were probed overnight at 4 °C with Inpp5f (1:500, cat# SAB2700848, Sigma, USA), p-Akt Ser473 (1:1000, cat# 9271, Cell signaling, USA), total Akt (1:1000, cat# 9272, Cell signaling, USA), p65 (1:1000, ab32536, Abcam, USA), Sp1 (1:1000, cat# 5931, Cell signaling, USA), β-actin (1:1000, cat# AB10024, Sangon, China), Lamin B (1:500, cat# BA1228, Boster, China) antibodies.

    Techniques: Expressing, Transfection

    siRNA -mediated depletion of SP1 or c-JUN expression in ER+ EC cells abolished TAM-stimulated promoter activity of TFF3 and growth in Matrigel culture (A) TFF3 promoter activity and (B) growth in Matrigel of EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. Depletion of SP1 or c-JUN expression was achieved using transient-transfection of si - RNA directed to SP1 or c-JUN transcript respectively, as described in materials and methods. Universal controls (scrambled oligo) were used for transfection control as described in materials and methods. The luciferase assay was performed as described in materials and methods. FM were 10%FBS, standard media conditions as per ATCC propagation instructions; and CSF-PRFM were charcoal striped 10% FBS, phenol-red free media. Statistical significance was assessed by using an unpaired two-tailed Student's t test ( P

    Journal: Oncotarget

    Article Title: Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells

    doi: 10.18632/oncotarget.20461

    Figure Lengend Snippet: siRNA -mediated depletion of SP1 or c-JUN expression in ER+ EC cells abolished TAM-stimulated promoter activity of TFF3 and growth in Matrigel culture (A) TFF3 promoter activity and (B) growth in Matrigel of EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. Depletion of SP1 or c-JUN expression was achieved using transient-transfection of si - RNA directed to SP1 or c-JUN transcript respectively, as described in materials and methods. Universal controls (scrambled oligo) were used for transfection control as described in materials and methods. The luciferase assay was performed as described in materials and methods. FM were 10%FBS, standard media conditions as per ATCC propagation instructions; and CSF-PRFM were charcoal striped 10% FBS, phenol-red free media. Statistical significance was assessed by using an unpaired two-tailed Student's t test ( P

    Article Snippet: IHC analysis was performed as previously described [ ] using rabbit anti-TFF3 was obtained from Abcam, Cambridge, MA; rabbit anti-SP1, rabbit anti-p-c-JUN and rabbit anti-c-JUN antibodies wereobtained from Cell Signaling Technology, Singapore [ ].

    Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Luciferase, Two Tailed Test

    TAM stimulates hypomethylation in the TFF3 promoter and modulates TFF3 transcription through c-JUN/SP1 (A) TFF3 promoter sequence (from -700 to +50bp) located on chromosome 21q22.3 (21:42316053-42314804). Thirteen CpG islands are positioned in the TFF3 promoter sequence, highlighted in red colour. Four binding sites for transcription factors, FOXA2 ( TATTTGATTTTA , -667 to -656bp), c-JUN ( TGTTTCA , -532 to -525bp; TGACTCA , -498 to -491bp), SP1 ( CACCACACCC , -379 to -369bp), and SOX5 ( CAAACAATCC , -119 to -110bp) were identified in the TFF3 promoter sequence, highlighted in bold font. Starting site, ATG, highlighted in green colour. Primer sites highlighted in black (P1) and grey (P2) colour. (B) PCR for bisulfite treated DNA . Total DNA was extracted from cells and treated with bisulfite as described in materials and methods. Sequence of unmethylated (U) and methylated (M) specific primers described in materials and methods. Primer specific sequence amplified on TFF3 promoter mentioned left side . The sizes of detected amplified product in base pair (bp) are shown on the right side. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. (C) TFF3 promoter activity in EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. The luciferase assay was performed as described in Materials and Methods. NT , negative control ( pGL3 basic construct ); PT , positive control ( pGL3-TFF3 construct ); pGL3-TFF3 construct with altered sequence of FOXA, c-JUN, SP1 , or SOX5 binding site (mentioned in Figure 2A ), respectively. Construct information is described in methodology section. (D) ChIP analysis in EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. ChIP assay was carried out using IgG (control) or SP1 antibody and c-JUN antibody and binding of SP1 and c-JUN was assessed using q-PCR as described in the methodology section. For each PCR, enrichment represent relative to the percentage of Input, error bars represent ±SD . FM were 10%FBS, standard media conditions as per ATCC propagation instructions; and CSF-PRFM were charcoal striped 10% FBS, phenol-red free media. Statistical significance was assessed by using an unpaired two-tailed Student's t test . Columns are mean of triplicate experiments; bars, ± SD . ** P

    Journal: Oncotarget

    Article Title: Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells

    doi: 10.18632/oncotarget.20461

    Figure Lengend Snippet: TAM stimulates hypomethylation in the TFF3 promoter and modulates TFF3 transcription through c-JUN/SP1 (A) TFF3 promoter sequence (from -700 to +50bp) located on chromosome 21q22.3 (21:42316053-42314804). Thirteen CpG islands are positioned in the TFF3 promoter sequence, highlighted in red colour. Four binding sites for transcription factors, FOXA2 ( TATTTGATTTTA , -667 to -656bp), c-JUN ( TGTTTCA , -532 to -525bp; TGACTCA , -498 to -491bp), SP1 ( CACCACACCC , -379 to -369bp), and SOX5 ( CAAACAATCC , -119 to -110bp) were identified in the TFF3 promoter sequence, highlighted in bold font. Starting site, ATG, highlighted in green colour. Primer sites highlighted in black (P1) and grey (P2) colour. (B) PCR for bisulfite treated DNA . Total DNA was extracted from cells and treated with bisulfite as described in materials and methods. Sequence of unmethylated (U) and methylated (M) specific primers described in materials and methods. Primer specific sequence amplified on TFF3 promoter mentioned left side . The sizes of detected amplified product in base pair (bp) are shown on the right side. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. (C) TFF3 promoter activity in EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. The luciferase assay was performed as described in Materials and Methods. NT , negative control ( pGL3 basic construct ); PT , positive control ( pGL3-TFF3 construct ); pGL3-TFF3 construct with altered sequence of FOXA, c-JUN, SP1 , or SOX5 binding site (mentioned in Figure 2A ), respectively. Construct information is described in methodology section. (D) ChIP analysis in EC cells. EC cells (VE or CTE) were cultured in FM or CSF-PRFM. 5μM TAM was used to treat cells. ChIP assay was carried out using IgG (control) or SP1 antibody and c-JUN antibody and binding of SP1 and c-JUN was assessed using q-PCR as described in the methodology section. For each PCR, enrichment represent relative to the percentage of Input, error bars represent ±SD . FM were 10%FBS, standard media conditions as per ATCC propagation instructions; and CSF-PRFM were charcoal striped 10% FBS, phenol-red free media. Statistical significance was assessed by using an unpaired two-tailed Student's t test . Columns are mean of triplicate experiments; bars, ± SD . ** P

    Article Snippet: IHC analysis was performed as previously described [ ] using rabbit anti-TFF3 was obtained from Abcam, Cambridge, MA; rabbit anti-SP1, rabbit anti-p-c-JUN and rabbit anti-c-JUN antibodies wereobtained from Cell Signaling Technology, Singapore [ ].

    Techniques: Sequencing, Binding Assay, Polymerase Chain Reaction, Methylation, Amplification, Cell Culture, Activity Assay, Luciferase, Negative Control, Construct, Positive Control, Chromatin Immunoprecipitation, Two Tailed Test

    MIR - 382 directly target SP1 in CRC. a The predicted MIR - 382 binding site within Sp2 3′-UTR and its mutated version. b MIR - 382 mimic inhibited the expression of SP1 in HT29 cells. c MIR - 382 mimic inhibited luciferase activity of cells transfected with pMIR-SP1-3′-UTR Wt but not pMIR-SP1-3′-UTR Mut. d Correlation between MIR - 382 and SP1 expression in CRC tissues. (***P

    Journal: Biological Research

    Article Title: MicroRNA-382 inhibits cell growth and migration in colorectal cancer by targeting SP1

    doi: 10.1186/s40659-018-0200-9

    Figure Lengend Snippet: MIR - 382 directly target SP1 in CRC. a The predicted MIR - 382 binding site within Sp2 3′-UTR and its mutated version. b MIR - 382 mimic inhibited the expression of SP1 in HT29 cells. c MIR - 382 mimic inhibited luciferase activity of cells transfected with pMIR-SP1-3′-UTR Wt but not pMIR-SP1-3′-UTR Mut. d Correlation between MIR - 382 and SP1 expression in CRC tissues. (***P

    Article Snippet: After blocked with 5% milk for 1 h, the membranes were incubated with the following antibodies: anti-SP1 rabbit antibody (#9389, Cell signaling Technology, Danvers, MA, USA); anti-E-Cadherin rabbit antibody (#3195, Cell signaling Technology); anti-N-Cadherin rabbit antibody (#13116, Cell signaling Technology); anti-Vimentin rabbit antibody (#5741, Cell signaling Technology); anti-Snail rabbit antibody (#3879, Cell signaling Technology); anti-GAPDH rabbit antibody (#5174, Cell signaling Technology).

    Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Transfection

    Knockdown of SP1 inhibits cell proliferation and migration. a The expression of SP1 in HT29 cell lines with synthetic siRNAs transfection. b CCK-8 assay was performed to measure cell proliferation in HT29 cell line with synthetic siRNAs or miRNAs transfection. c Wound-healing assay was performed to measure cell migration in HT29 cell line with synthetic siRNAs or miRNAs transfection. d Western blot was conducted to measure the expression of E-Cadherin, N-Cadherin, Vimentin, and Snail. (***P

    Journal: Biological Research

    Article Title: MicroRNA-382 inhibits cell growth and migration in colorectal cancer by targeting SP1

    doi: 10.1186/s40659-018-0200-9

    Figure Lengend Snippet: Knockdown of SP1 inhibits cell proliferation and migration. a The expression of SP1 in HT29 cell lines with synthetic siRNAs transfection. b CCK-8 assay was performed to measure cell proliferation in HT29 cell line with synthetic siRNAs or miRNAs transfection. c Wound-healing assay was performed to measure cell migration in HT29 cell line with synthetic siRNAs or miRNAs transfection. d Western blot was conducted to measure the expression of E-Cadherin, N-Cadherin, Vimentin, and Snail. (***P

    Article Snippet: After blocked with 5% milk for 1 h, the membranes were incubated with the following antibodies: anti-SP1 rabbit antibody (#9389, Cell signaling Technology, Danvers, MA, USA); anti-E-Cadherin rabbit antibody (#3195, Cell signaling Technology); anti-N-Cadherin rabbit antibody (#13116, Cell signaling Technology); anti-Vimentin rabbit antibody (#5741, Cell signaling Technology); anti-Snail rabbit antibody (#3879, Cell signaling Technology); anti-GAPDH rabbit antibody (#5174, Cell signaling Technology).

    Techniques: Migration, Expressing, Transfection, CCK-8 Assay, Wound Healing Assay, Western Blot

    Therapeutic targeting of HBP in CRPC. ( a ) Decreased HBP expression in CRPC tumours containing AR-FL is associated with increased activity of PI3K-AKT that could activate AR. This activates cell cycle genes leading to increased proliferation driving CRPC progression. ( b ) In CRPC containing AR-V7, decreased HBP expression activates SP1-regulated ChREBP expression leading to stimulation of cell cycle genes, increased proliferation and tumour progression. ( c ) MTT assay results for LNCaP-ABL ( n =9) cells treated with vehicle (PBS+dimethylsulphoxide) or 10 μM enzalutamide (Enz) or 60 mM UDP-N-acetylglucosamine (UDP-GlcNAc) or both enz+UDP-GlcNAc for 96 h. ( d ) Same as in c but treatments were done on 22Rv1 cells ( n =8). In both c and d , differences in the level of mean absorbance with corresponding confidence interval (CI) to control obtained for each of the conditions ( x axis) is represented on the y axis. Comparisons of all treatments with control group in c , d were significant at a P -value of 0.01 (Bonferroni corrected). Pairwise comparisons of either Enz or combination of Enz+UDP-GlcNAc with other treatments were significant at a P -value

    Journal: Nature Communications

    Article Title: Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer

    doi: 10.1038/ncomms11612

    Figure Lengend Snippet: Therapeutic targeting of HBP in CRPC. ( a ) Decreased HBP expression in CRPC tumours containing AR-FL is associated with increased activity of PI3K-AKT that could activate AR. This activates cell cycle genes leading to increased proliferation driving CRPC progression. ( b ) In CRPC containing AR-V7, decreased HBP expression activates SP1-regulated ChREBP expression leading to stimulation of cell cycle genes, increased proliferation and tumour progression. ( c ) MTT assay results for LNCaP-ABL ( n =9) cells treated with vehicle (PBS+dimethylsulphoxide) or 10 μM enzalutamide (Enz) or 60 mM UDP-N-acetylglucosamine (UDP-GlcNAc) or both enz+UDP-GlcNAc for 96 h. ( d ) Same as in c but treatments were done on 22Rv1 cells ( n =8). In both c and d , differences in the level of mean absorbance with corresponding confidence interval (CI) to control obtained for each of the conditions ( x axis) is represented on the y axis. Comparisons of all treatments with control group in c , d were significant at a P -value of 0.01 (Bonferroni corrected). Pairwise comparisons of either Enz or combination of Enz+UDP-GlcNAc with other treatments were significant at a P -value

    Article Snippet: Immunoprecipitation was carried out using either 10 μg of rabbit SP1 antibody (Cell Signaling) or using rabbit non-immune IgG (Sigma), as a control.

    Techniques: Expressing, Activity Assay, MTT Assay

    EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Labeling, Incubation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Polymerase Chain Reaction

    Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Construct, Plasmid Preparation, Transfection

    Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Western Blot, In Vivo, In Vitro, Expressing

    Western Blot analysis of β-catenin expression in RMS tumor cell lines. (A) Western Blot analysis of ß-catenin expression in various RMS tumor cell lines. ß-actin served as loading control. (B) Western Blot analysis of ß-catenin expression in three different RMS tumor cell lines after 48 h treatment with either FH535, XAV939, ß-catenin siRNA, or (C) WNT3A treatment. ß-actin served as loading control. (D) Densitometric quantification of the Western Blots shown in (C) using ImageJ. Expression of ß-catenin after treatment (+) was normalized to ß-actin and compared to untreated cells (−). (E) subcellular localization of ß-catenin in the RMS tumor cell lines with and without WNT3A stimulation. GAPDH served as cytoplasmatic and SP1 as nuclear marker to check purity of the protein isolation from different compartments. CP, cytoplasmatic; N, nuclear.

    Journal: Frontiers in Pediatrics

    Article Title: Canonical WNT/β-Catenin Signaling Plays a Subordinate Role in Rhabdomyosarcomas

    doi: 10.3389/fped.2018.00378

    Figure Lengend Snippet: Western Blot analysis of β-catenin expression in RMS tumor cell lines. (A) Western Blot analysis of ß-catenin expression in various RMS tumor cell lines. ß-actin served as loading control. (B) Western Blot analysis of ß-catenin expression in three different RMS tumor cell lines after 48 h treatment with either FH535, XAV939, ß-catenin siRNA, or (C) WNT3A treatment. ß-actin served as loading control. (D) Densitometric quantification of the Western Blots shown in (C) using ImageJ. Expression of ß-catenin after treatment (+) was normalized to ß-actin and compared to untreated cells (−). (E) subcellular localization of ß-catenin in the RMS tumor cell lines with and without WNT3A stimulation. GAPDH served as cytoplasmatic and SP1 as nuclear marker to check purity of the protein isolation from different compartments. CP, cytoplasmatic; N, nuclear.

    Article Snippet: For analysis of the subcellular localization, the following antibodies were used: monoclonal rabbit anti-human β-catenin (Cell Signaling Technology (CST), Danvers, MA, USA), non-phospho (active) monoclonal rabbit anti-human β-catenin (CST), monoclonal rabbit anti-human SP1 (CST), and monoclonal mouse anti-human GAPH.

    Techniques: Western Blot, Expressing, Marker, Isolation

    SP1 is a direct target gene of miR-411 in vitro . (A) TargetScan revealed that SP1 mRNA contained an miR-411 seed match at position 3,325–3,331 of the SP1 3′UTR. (B) Western blot analysis revealed that SP1 was significantly downregulated in MCF-7 and SKBR3 cells following transfection with miR-411. (C) Overexpression of miR-411 significantly inhibited the luciferase activity of SP1 Wt, but not SP1 Mut in the MCF-7 and SKBR3 cells. Data are presented as the mean ± standard deviation. * P

    Journal: Molecular Medicine Reports

    Article Title: miRNA-411 acts as a potential tumor suppressor miRNA via the downregulation of specificity protein 1 in breast cancer

    doi: 10.3892/mmr.2016.5645

    Figure Lengend Snippet: SP1 is a direct target gene of miR-411 in vitro . (A) TargetScan revealed that SP1 mRNA contained an miR-411 seed match at position 3,325–3,331 of the SP1 3′UTR. (B) Western blot analysis revealed that SP1 was significantly downregulated in MCF-7 and SKBR3 cells following transfection with miR-411. (C) Overexpression of miR-411 significantly inhibited the luciferase activity of SP1 Wt, but not SP1 Mut in the MCF-7 and SKBR3 cells. Data are presented as the mean ± standard deviation. * P

    Article Snippet: Western blot analysis The rabbit anti-human monoclonal SP1 (1:1,000; cat. no. 9389) and rabbit anti-human monoclonal GADPH (1:1,000; cat. no. 2118) primary antibodies used in the present study were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: In Vitro, Western Blot, Transfection, Over Expression, Luciferase, Activity Assay, Standard Deviation

    Western blot analysis of Sp1 protein in 2DG-treated NCCIT cells. The whole cell lysates of NCCIT cells treated with 2DG for the indicated times (0, 24, 72, or 168 h) were precipitated with an anti-Sp1 antibody (D4C3), and the precipitated proteins were immunoblotted with D4C3, anti- O -GlcNAc antibodies (RL2 or CTD110.6), anti-Phosphoserine/threonine (P-Ser/Thr), anti-SUMO1, or anti-Ubiquitin. These transcriptional modifications were observed in Sp1 proteins [6] . Among these modifications, only O -GlcNAcylation was clearly detected in the D4C3 precipitates. The Sp1 levels in whole cell lysates were shown as a reference (left panel).

    Journal: Data in Brief

    Article Title: Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    doi: 10.1016/j.dib.2016.12.001

    Figure Lengend Snippet: Western blot analysis of Sp1 protein in 2DG-treated NCCIT cells. The whole cell lysates of NCCIT cells treated with 2DG for the indicated times (0, 24, 72, or 168 h) were precipitated with an anti-Sp1 antibody (D4C3), and the precipitated proteins were immunoblotted with D4C3, anti- O -GlcNAc antibodies (RL2 or CTD110.6), anti-Phosphoserine/threonine (P-Ser/Thr), anti-SUMO1, or anti-Ubiquitin. These transcriptional modifications were observed in Sp1 proteins [6] . Among these modifications, only O -GlcNAcylation was clearly detected in the D4C3 precipitates. The Sp1 levels in whole cell lysates were shown as a reference (left panel).

    Article Snippet: The primary antibodies used were mouse anti-O -GlcNAc monoclonal antibodies (RL2, Thermo Fisher Scientific, Waltham, MA; CTD110.6, Cell Signaling Technology), rabbit anti-Sp1 monoclonal antibody (D4C3, Cell Signaling Technology), rabbit anti-GRP78/Bip monoclonal antibody (C50B12, Cell Signaling Technology), rabbit anti-GAPDH monoclonal antibody (D16H11, Cell Signaling Technology), rabbit anti-β-Actin polyclonal antibody (#4967, Cell Signaling Technology), rabbit anti-Phosphoserine/threonine polyclonal antibody (ab17464, abcam, Cambridge, UK), mouse anti-SUMO1 monoclonal antibody (21C7, BostonBiochem, Cambridge, MA), and mouse anti-Ubiquitin monoclonal antibody (P4D1, Cell Signaling Technology).

    Techniques: Western Blot

    ZEB1 activates VEGFA transcription by recruiting SP1 to the endogenous VEGFA promoter. (A) Sequential deletion and mutation of SP1 elements on the human VEGFA promoter were fused to the luciferase reporter. (B) MDA-MB-231 cells were co-transfected with the ZEB1 expression plasmid (1 μg/well) and different wild-type VEGFA promoter luciferase reporter constructs (1 μg/well). Extract luciferase activities were determined 48 h after transfection using a Betascope analyzer. Luciferase values were normalized to Renilla activities. * P

    Journal: PLoS ONE

    Article Title: ZEB1 Upregulates VEGF Expression and Stimulates Angiogenesis in Breast Cancer

    doi: 10.1371/journal.pone.0148774

    Figure Lengend Snippet: ZEB1 activates VEGFA transcription by recruiting SP1 to the endogenous VEGFA promoter. (A) Sequential deletion and mutation of SP1 elements on the human VEGFA promoter were fused to the luciferase reporter. (B) MDA-MB-231 cells were co-transfected with the ZEB1 expression plasmid (1 μg/well) and different wild-type VEGFA promoter luciferase reporter constructs (1 μg/well). Extract luciferase activities were determined 48 h after transfection using a Betascope analyzer. Luciferase values were normalized to Renilla activities. * P

    Article Snippet: The Abs used in these experiments were rabbit monoclonal Ab against SP1 (#9389S; CST) and anti-rabbit normal IgG (sc-2345, Santa Cruz).

    Techniques: Mutagenesis, Luciferase, Multiple Displacement Amplification, Transfection, Expressing, Plasmid Preparation, Construct