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  • 99
    Name:
    SP1 Antibody
    Description:
    Specificity protein 1 SP1 is a ubiquitously expressed transcription factor belonging to the family of C2H2 type zinc finger containing DNA binding proteins SP1 binds GC rich motifs with high affinity and regulates the expression of numerous mammalian genes 1 2 It interacts with many other transcription factors such as c Myc EGR1 and Stat1 and with basal transcription machinery components SP1 interacts with chromatin modifying factors such as histone deacetylases HDACs and p300 in chromatin remodeling Transcriptional activity and stability of SP1 are regulated by post translational modification including phosphorylation acetylation ubiquitination and glycosylation 3 Glycosylation of SP1 following insulin treatment leads to increased nuclear localization while glucagon treatment increases cytoplasmic SP1 levels 4 6 Investigators have found high levels of SP1 in patients with Alzheimer s disease 7
    Catalog Number:
    5931
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro593 of human SP1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti sp1
    <t>Sp1</t> downregulation causes tumor regression. MIA PaCa-2 cells injected subcutaneously, and treated with saline or mithramycin for 4 weeks. ( A ) Tumors explanted from saline, mithramycin 0.3 mg/kg, and mithramycin 0.6 mg/kg ( B ) Tumor volume ( C ) Tumor weights ( D ) Tumors analyzed for apoptosis with cleaved PARP by western blot, and ( E ) cleaved caspase 3 by immunofluorescence.
    Specificity protein 1 SP1 is a ubiquitously expressed transcription factor belonging to the family of C2H2 type zinc finger containing DNA binding proteins SP1 binds GC rich motifs with high affinity and regulates the expression of numerous mammalian genes 1 2 It interacts with many other transcription factors such as c Myc EGR1 and Stat1 and with basal transcription machinery components SP1 interacts with chromatin modifying factors such as histone deacetylases HDACs and p300 in chromatin remodeling Transcriptional activity and stability of SP1 are regulated by post translational modification including phosphorylation acetylation ubiquitination and glycosylation 3 Glycosylation of SP1 following insulin treatment leads to increased nuclear localization while glucagon treatment increases cytoplasmic SP1 levels 4 6 Investigators have found high levels of SP1 in patients with Alzheimer s disease 7
    https://www.bioz.com/result/anti sp1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    anti sp1 - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Inhibition of Sp1 prevents ER homeostasis and causes cell death by lysosomal membrane permeabilization in pancreatic cancer"

    Article Title: Inhibition of Sp1 prevents ER homeostasis and causes cell death by lysosomal membrane permeabilization in pancreatic cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01696-2

    Sp1 downregulation causes tumor regression. MIA PaCa-2 cells injected subcutaneously, and treated with saline or mithramycin for 4 weeks. ( A ) Tumors explanted from saline, mithramycin 0.3 mg/kg, and mithramycin 0.6 mg/kg ( B ) Tumor volume ( C ) Tumor weights ( D ) Tumors analyzed for apoptosis with cleaved PARP by western blot, and ( E ) cleaved caspase 3 by immunofluorescence.
    Figure Legend Snippet: Sp1 downregulation causes tumor regression. MIA PaCa-2 cells injected subcutaneously, and treated with saline or mithramycin for 4 weeks. ( A ) Tumors explanted from saline, mithramycin 0.3 mg/kg, and mithramycin 0.6 mg/kg ( B ) Tumor volume ( C ) Tumor weights ( D ) Tumors analyzed for apoptosis with cleaved PARP by western blot, and ( E ) cleaved caspase 3 by immunofluorescence.

    Techniques Used: Injection, Western Blot, Immunofluorescence

    Schematic diagram showing how Sp1 downregulation prevents ER homeostasis.
    Figure Legend Snippet: Schematic diagram showing how Sp1 downregulation prevents ER homeostasis.

    Techniques Used:

    Sp1 downregulation leads to chronic ER stress by deregulating the homeostatic mechanism. Mithramycin treated MIA PaCa-2 cells analyzed for ( A ) ERSE dual-luciferase activity assay from 0–24 hours, and ( B ) ChIP of Sp1 on the Grp78 promoter, from 0–24 hours.
    Figure Legend Snippet: Sp1 downregulation leads to chronic ER stress by deregulating the homeostatic mechanism. Mithramycin treated MIA PaCa-2 cells analyzed for ( A ) ERSE dual-luciferase activity assay from 0–24 hours, and ( B ) ChIP of Sp1 on the Grp78 promoter, from 0–24 hours.

    Techniques Used: Luciferase, Activity Assay, Chromatin Immunoprecipitation

    2) Product Images from "TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b"

    Article Title: TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b

    Journal: Cell Cycle

    doi: 10.1080/15384101.2018.1556063

    TGF-β1 promoted the reparation of cartilage tissues in vivo . Rats were divided into OA+MSC-exosome group (rats received articular cavity injection of MSC-exosome, 100 μl; 1 × 10 11 MSC-exosome particles/ml; n = 6), OA+TGF-β1+ MSC-exosome group (rats received articular cavity injection of TGF-β1-exosome, 100 μl; 1 × 10 11 TGF-β1-exosome particles/ml; n = 6), OA+TGF-β1+ MSC-NC-exosome group (rats received articular cavity injection of TGF-β1-NC-exosome, 100 μl; 1 × 10 11 TGF-β1-NC-exosome particles/ml; n = 6), and OA+TGF-β1+ MSC-miR135b inhibitor-exosome group (rats received articular cavity injection of TGF-β1-miR135b inhibitor-exosome, 100 μl; 1 × 10 11 TGF-β1-miR135b inhibitor-exosome particles/ml; n = 6). Twelve weeks after surgery, rats were sacrificed and the knee samples were harvested to evaluate disease progression. (A) OARSI score. (B) Chondrocytes count. (C) MiR-135b expressions in cartilage. (D) Sp1 expression in cartilage. * P
    Figure Legend Snippet: TGF-β1 promoted the reparation of cartilage tissues in vivo . Rats were divided into OA+MSC-exosome group (rats received articular cavity injection of MSC-exosome, 100 μl; 1 × 10 11 MSC-exosome particles/ml; n = 6), OA+TGF-β1+ MSC-exosome group (rats received articular cavity injection of TGF-β1-exosome, 100 μl; 1 × 10 11 TGF-β1-exosome particles/ml; n = 6), OA+TGF-β1+ MSC-NC-exosome group (rats received articular cavity injection of TGF-β1-NC-exosome, 100 μl; 1 × 10 11 TGF-β1-NC-exosome particles/ml; n = 6), and OA+TGF-β1+ MSC-miR135b inhibitor-exosome group (rats received articular cavity injection of TGF-β1-miR135b inhibitor-exosome, 100 μl; 1 × 10 11 TGF-β1-miR135b inhibitor-exosome particles/ml; n = 6). Twelve weeks after surgery, rats were sacrificed and the knee samples were harvested to evaluate disease progression. (A) OARSI score. (B) Chondrocytes count. (C) MiR-135b expressions in cartilage. (D) Sp1 expression in cartilage. * P

    Techniques Used: In Vivo, Injection, Expressing

    MiR-135b negatively regulated Sp1. (a) The binding site between miR-135b and Sp1 3ʹUTR. (b) The luciferase activity of Sp1 3ʹUTR in HEK293 cells co-transfected with miR-135b mimics or miR-135b inhibitor. (c) The protein expression of Sp1 in C5.18 cells. Pre-NC was a control of miR-135b mimics; NC was a control of miR-135b inhibitor. * P
    Figure Legend Snippet: MiR-135b negatively regulated Sp1. (a) The binding site between miR-135b and Sp1 3ʹUTR. (b) The luciferase activity of Sp1 3ʹUTR in HEK293 cells co-transfected with miR-135b mimics or miR-135b inhibitor. (c) The protein expression of Sp1 in C5.18 cells. Pre-NC was a control of miR-135b mimics; NC was a control of miR-135b inhibitor. * P

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

    ). MSC-exosomes were isolated from the 2 groups. C5.18 cells were divided into TGF-β1+ NC+si-control group (after transfected with si-control, C5.18 cells were co-cultured with TGF-β1-exosome), TGF-β1+ miR-135b inhibitor+si-control group (after transfected with si-control, C5.18 cells were co-cultured with TGF-β1+ miR-135b inhibitor-exosome), TGF-β1+ NC+si-Sp1 group (after transfected with si-Sp1, C5.18 cells were co-cultured with TGF-β1-exosome), and TGF-β1+ miR-135b inhibitor+si-Sp1 group (after transfected with si-Sp1, C5.18 cells were co-cultured with TGF-β1+ miR-135b inhibitor-exosome). (a) Sp1 expression in C5.18 cells. (b) The cell viability of C5.18 cells. * P
    Figure Legend Snippet: ). MSC-exosomes were isolated from the 2 groups. C5.18 cells were divided into TGF-β1+ NC+si-control group (after transfected with si-control, C5.18 cells were co-cultured with TGF-β1-exosome), TGF-β1+ miR-135b inhibitor+si-control group (after transfected with si-control, C5.18 cells were co-cultured with TGF-β1+ miR-135b inhibitor-exosome), TGF-β1+ NC+si-Sp1 group (after transfected with si-Sp1, C5.18 cells were co-cultured with TGF-β1-exosome), and TGF-β1+ miR-135b inhibitor+si-Sp1 group (after transfected with si-Sp1, C5.18 cells were co-cultured with TGF-β1+ miR-135b inhibitor-exosome). (a) Sp1 expression in C5.18 cells. (b) The cell viability of C5.18 cells. * P

    Techniques Used: Isolation, Transfection, Cell Culture, Expressing

    3) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

    Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096512

    Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.
    Figure Legend Snippet: Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Generated, Cell Culture, Western Blot

    Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
    Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p

    Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 participates in the Aldh1a2 expression. ( A ) The genomic organization of the mouse Aldh1a2 gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR ( Left panel ), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting ( Right panel ). Relative mRNA expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 ( Left panel ). Data are representative of three ( Left panel ) or two ( Right panel ) independent experiments. ( C ) Serial-deletion fragments derived from the 5′-flanking region of the mouse Aldh1a2 gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (** p
    Figure Legend Snippet: Sp1 participates in the Aldh1a2 expression. ( A ) The genomic organization of the mouse Aldh1a2 gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR ( Left panel ), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting ( Right panel ). Relative mRNA expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 ( Left panel ). Data are representative of three ( Left panel ) or two ( Right panel ) independent experiments. ( C ) Serial-deletion fragments derived from the 5′-flanking region of the mouse Aldh1a2 gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (** p

    Techniques Used: Expressing, Binding Assay, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Derivative Assay, Plasmid Preparation, Transfection, Construct, Luciferase, Activity Assay

    The MEK1-ERK-signaling pathway and the p38 MAPK-signaling pathway are required for the GM-CSF-induced Aldh1a2 expression and nuclear translocation of Sp1 in BM-DCs. ( A ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF for 16 h in the presence or absence of 50 µM PD98059 (PD) or 25 µM SB203580 (SB). After the culture, Aldh1a2 gene expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. The results are shown as the mean + SD of triplicate cultures. Statistical significance was determined by the Student's t test (*** p
    Figure Legend Snippet: The MEK1-ERK-signaling pathway and the p38 MAPK-signaling pathway are required for the GM-CSF-induced Aldh1a2 expression and nuclear translocation of Sp1 in BM-DCs. ( A ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF for 16 h in the presence or absence of 50 µM PD98059 (PD) or 25 µM SB203580 (SB). After the culture, Aldh1a2 gene expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. The results are shown as the mean + SD of triplicate cultures. Statistical significance was determined by the Student's t test (*** p

    Techniques Used: Expressing, Translocation Assay, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p
    Figure Legend Snippet: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p

    Techniques Used: Binding Assay, Activity Assay, Transfection, Sequencing, Plasmid Preparation, Expressing, Luciferase, Generated, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    4) Product Images from "Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression, et al. Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression"

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

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14663

    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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

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

    5) Product Images from "SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis"

    Article Title: SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0962-6

    The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P

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

    6) Product Images from "ETS1 and SP1 drive DHX15 expression in acute lymphoblastic leukaemia, et al. ETS1 and SP1 drive DHX15 expression in acute lymphoblastic leukaemia"

    Article Title: ETS1 and SP1 drive DHX15 expression in acute lymphoblastic leukaemia, et al. ETS1 and SP1 drive DHX15 expression in acute lymphoblastic leukaemia

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13525

    EMSA and Ch IP analyses of ETS 1 and SP 1 binding to the DHX 15 promoter. A, EMSA of ETS1 (left) and SP1 (right). The 5′‐biotin end‐labelled probe was incubated in the absence (lane 0) or presence (lane 1) of nuclear extracts from Jurkat and NALM6 cells. A cold mutated probe (lane 2) and cold probe (lane 3) were used as competitors at concentrations that were in a 100‐fold molar excess to the biotin‐labelled probe. Supershift assays were performed with 4 μg of a specific antibody against ETS1 or SP1 (lane 4). B, Equal amounts of Jurkat and NALM6 chromatin were immunoprecipitated with antibodies for ETS1 and SP1 and subsequently quantified through agarose gel electrophoresis using a primer set specific for the basal region (−181 to −36 bp). Moreover, immunoprecipitated DNA was amplified using a primer set specific to the off‐target region (GAPDH) shown in the lower panel as a negative control
    Figure Legend Snippet: EMSA and Ch IP analyses of ETS 1 and SP 1 binding to the DHX 15 promoter. A, EMSA of ETS1 (left) and SP1 (right). The 5′‐biotin end‐labelled probe was incubated in the absence (lane 0) or presence (lane 1) of nuclear extracts from Jurkat and NALM6 cells. A cold mutated probe (lane 2) and cold probe (lane 3) were used as competitors at concentrations that were in a 100‐fold molar excess to the biotin‐labelled probe. Supershift assays were performed with 4 μg of a specific antibody against ETS1 or SP1 (lane 4). B, Equal amounts of Jurkat and NALM6 chromatin were immunoprecipitated with antibodies for ETS1 and SP1 and subsequently quantified through agarose gel electrophoresis using a primer set specific for the basal region (−181 to −36 bp). Moreover, immunoprecipitated DNA was amplified using a primer set specific to the off‐target region (GAPDH) shown in the lower panel as a negative control

    Techniques Used: Binding Assay, Incubation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Negative Control

    7) Product Images from "The human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation"

    Article Title: The human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00098.2018

    Depletion of TRPM2 followed by doxorubicin (Doxo) exposure reduces phosphorylated Src, phosphorylated CREB, and total CREB but increases total Src in the nucleus. A : TRPM2-depleted and scrambled SH-SY5Y control cells were treated for 24 or 48 h with Doxo and then fractionated into cytosolic and nuclear fractions. A representative Western blot from one of two experiments is shown. Densitometry measurements of nuclear protein for two experiments, each using two different clones from each group ( n = 4), were standardized to results for each experiment’s scrambled nuclear control at time 0 . The means ± SE of phosphorylated or total Src or CREB calculated are shown. Western blotting of nuclear fractions revealed that pSrc (group × exposure time interaction effect, P = 0.0002), pCREB (group × exposure time interaction effect, P = 0.0457), and total CREB (group × exposure time interaction effect, P = 0.019) were significantly reduced in the nucleus of TRPM2-depleted cells after Doxo compared with scrambled controls cells. Nuclear Src was significantly increased (group × exposure time interaction effect, P = 0.05) in TRPM2 depleted cells after doxorubicin treatment compared with control scrambled cells. * P ≤ 0.05, group × Doxo exposure time interaction effect analyzed with two-way ANOVA. B : quality of fractionation was determined by probing cytosolic (C), membrane (M), and nuclear (N) fractions with antibody to GAPDH, EGFR, and SP1, respectively. CREB, cAMP-responsive element-binding protein; EGFR, epidermal growth factor receptor; KO, knockout; MCU, mitochondrial calcium uniporter; p, phosphorylated; SP1, specificity protein 1; TRPM2, transient receptor potential melastatin channel subfamily member 2.
    Figure Legend Snippet: Depletion of TRPM2 followed by doxorubicin (Doxo) exposure reduces phosphorylated Src, phosphorylated CREB, and total CREB but increases total Src in the nucleus. A : TRPM2-depleted and scrambled SH-SY5Y control cells were treated for 24 or 48 h with Doxo and then fractionated into cytosolic and nuclear fractions. A representative Western blot from one of two experiments is shown. Densitometry measurements of nuclear protein for two experiments, each using two different clones from each group ( n = 4), were standardized to results for each experiment’s scrambled nuclear control at time 0 . The means ± SE of phosphorylated or total Src or CREB calculated are shown. Western blotting of nuclear fractions revealed that pSrc (group × exposure time interaction effect, P = 0.0002), pCREB (group × exposure time interaction effect, P = 0.0457), and total CREB (group × exposure time interaction effect, P = 0.019) were significantly reduced in the nucleus of TRPM2-depleted cells after Doxo compared with scrambled controls cells. Nuclear Src was significantly increased (group × exposure time interaction effect, P = 0.05) in TRPM2 depleted cells after doxorubicin treatment compared with control scrambled cells. * P ≤ 0.05, group × Doxo exposure time interaction effect analyzed with two-way ANOVA. B : quality of fractionation was determined by probing cytosolic (C), membrane (M), and nuclear (N) fractions with antibody to GAPDH, EGFR, and SP1, respectively. CREB, cAMP-responsive element-binding protein; EGFR, epidermal growth factor receptor; KO, knockout; MCU, mitochondrial calcium uniporter; p, phosphorylated; SP1, specificity protein 1; TRPM2, transient receptor potential melastatin channel subfamily member 2.

    Techniques Used: Western Blot, Clone Assay, Fractionation, Binding Assay, Knock-Out

    8) Product Images from "JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis"

    Article Title: JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01617-8

    Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway
    Figure Legend Snippet: Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway

    Techniques Used: Immunohistochemistry, Expressing

    JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P
    Figure Legend Snippet: JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P

    Techniques Used: Western Blot, Transfection, Tube Formation Assay

    JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P
    Figure Legend Snippet: JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P

    Techniques Used: Modification, Western Blot, Transfection

    9) Product Images from "miR-4721, Induced by EBV-miR-BART22, Targets GSK3β to Enhance the Tumorigenic Capacity of NPC through the WNT/β-catenin Pathway"

    Article Title: miR-4721, Induced by EBV-miR-BART22, Targets GSK3β to Enhance the Tumorigenic Capacity of NPC through the WNT/β-catenin Pathway

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2020.09.021

    Sp1 Binds to the Promoter Region of miR-4721, and EBV-miR-BART22 Induces miR-4721 through PI3K / AKT / c-JUN / Sp1 (A) Schematic representation of the promoter regions of miR-4721 with the putative Sp1 binding sites (site A, site B, and site C). (B) miR-4721 expression was detected by qPCR after Sp1 plasmid was transfected in both HONE1 and SUNE1 cells. Student’s t test. Mean ± SD. ∗∗∗p
    Figure Legend Snippet: Sp1 Binds to the Promoter Region of miR-4721, and EBV-miR-BART22 Induces miR-4721 through PI3K / AKT / c-JUN / Sp1 (A) Schematic representation of the promoter regions of miR-4721 with the putative Sp1 binding sites (site A, site B, and site C). (B) miR-4721 expression was detected by qPCR after Sp1 plasmid was transfected in both HONE1 and SUNE1 cells. Student’s t test. Mean ± SD. ∗∗∗p

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection

    10) Product Images from "miR-4721, Induced by EBV-miR-BART22, Targets GSK3β to Enhance the Tumorigenic Capacity of NPC through the WNT/β-catenin Pathway"

    Article Title: miR-4721, Induced by EBV-miR-BART22, Targets GSK3β to Enhance the Tumorigenic Capacity of NPC through the WNT/β-catenin Pathway

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2020.09.021

    Sp1 Binds to the Promoter Region of miR-4721, and EBV-miR-BART22 Induces miR-4721 through PI3K / AKT / c-JUN / Sp1 (A) Schematic representation of the promoter regions of miR-4721 with the putative Sp1 binding sites (site A, site B, and site C). (B) miR-4721 expression was detected by qPCR after Sp1 plasmid was transfected in both HONE1 and SUNE1 cells. Student’s t test. Mean ± SD. ∗∗∗p
    Figure Legend Snippet: Sp1 Binds to the Promoter Region of miR-4721, and EBV-miR-BART22 Induces miR-4721 through PI3K / AKT / c-JUN / Sp1 (A) Schematic representation of the promoter regions of miR-4721 with the putative Sp1 binding sites (site A, site B, and site C). (B) miR-4721 expression was detected by qPCR after Sp1 plasmid was transfected in both HONE1 and SUNE1 cells. Student’s t test. Mean ± SD. ∗∗∗p

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection

    11) Product Images from "Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11700

    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
    Figure Legend 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

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

    12) Product Images from "ERK3 promotes endothelial cell functions by upregulating SRC-3/SP1-mediated VEGFR2 expression"

    Article Title: ERK3 promotes endothelial cell functions by upregulating SRC-3/SP1-mediated VEGFR2 expression

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.24596

    ERK3 and SRC-3 synergistically regulate SP1-mediated VEGFR2 gene promoter activity (A ). HUVEC cells were transfected with SRC-3 siRNA (SRC-3si) or the non-targeting negative control siRNA (Ctrlsi). Expression levels of SRC-3 and VEGFR2 were analyzed by Western blotting. ( B ). A schematic illustration of a VEGFR2 gene promoter-driven luciferase reporter construct (pGL3-VEGFR2-Luc). A SP1 binding element resides in the VEGFR2 gene promoter. ( C ) and ( D ). ERK3, SRC-3 and SP1 synergistically promote VEGFR2 promoter-driven luciferase activity. HUVECs were transfected with pGL3-VEGFR2-Luc and SRC-3, ERK3, or SP1 construct as indicated. Luciferase activity is represented as relative luciferase units (RLU). ( E ). Knockdown of SRC-3 diminished the stimulatory effect of ERK3 and SP1 on VEGFR2 gene promoter-driven luciferase activity. HUVECs were transfected with pGL3-VEGFR2-Luc and ERK3 or SP1 construct, together with either SRC-3 siRNA (SRC-3si) or non-targeting control siRNA (Ctrlsi) as indicated.
    Figure Legend Snippet: ERK3 and SRC-3 synergistically regulate SP1-mediated VEGFR2 gene promoter activity (A ). HUVEC cells were transfected with SRC-3 siRNA (SRC-3si) or the non-targeting negative control siRNA (Ctrlsi). Expression levels of SRC-3 and VEGFR2 were analyzed by Western blotting. ( B ). A schematic illustration of a VEGFR2 gene promoter-driven luciferase reporter construct (pGL3-VEGFR2-Luc). A SP1 binding element resides in the VEGFR2 gene promoter. ( C ) and ( D ). ERK3, SRC-3 and SP1 synergistically promote VEGFR2 promoter-driven luciferase activity. HUVECs were transfected with pGL3-VEGFR2-Luc and SRC-3, ERK3, or SP1 construct as indicated. Luciferase activity is represented as relative luciferase units (RLU). ( E ). Knockdown of SRC-3 diminished the stimulatory effect of ERK3 and SP1 on VEGFR2 gene promoter-driven luciferase activity. HUVECs were transfected with pGL3-VEGFR2-Luc and ERK3 or SP1 construct, together with either SRC-3 siRNA (SRC-3si) or non-targeting control siRNA (Ctrlsi) as indicated.

    Techniques Used: Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Luciferase, Construct, Binding Assay

    ERK3 enhances the interactions of SRC3 with CBP and SP1 (A ) and ( B ). Cells were co-transfected with SRC-3Flag and ERK3 or the corresponding empty vector. The interactions of SRC-3 with CBP and SP1 were analyzed by immunoprecipitation (IP) using a Flag Ab or a mouse IgG, followed by Western blotting. The relative SRC-3 binding capacity with CBP or SP1 was determined by measuring the CBP or SP1 immunoblot (IB) band intensity in the IP samples. The band intensity in control vector group was arbitrarily set as “1.0”. Overexpression of ERK3 ( A ) enhanced the interactions of SRC-3 with CBP and SP1 shown in ( B ). (C ) and ( D ). Cells were transfected with ERK3 siRNA (ERK3si) or the silencer negative control siRNA (Ctrlsi), together with SRC-3Flag plasmid. The interactions of SRC-3 with CBP and SP1 were analyzed by IP using a Flag Ab, followed by Western blotting. Numbers below the immunoblots of CBP and SP1 in Flag Ab/IP samples represent the relative SRC-3 binding capacity with these proteins, which is determined by the ratio of the band intensity in Flag Ab/IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity (the ratio) in Ctrlsi group was arbitrarily set as “1.0”. Knockdown of ERK3 ( C ) greatly decreased the association of SRC3 with CBP and SP1 ( D ).
    Figure Legend Snippet: ERK3 enhances the interactions of SRC3 with CBP and SP1 (A ) and ( B ). Cells were co-transfected with SRC-3Flag and ERK3 or the corresponding empty vector. The interactions of SRC-3 with CBP and SP1 were analyzed by immunoprecipitation (IP) using a Flag Ab or a mouse IgG, followed by Western blotting. The relative SRC-3 binding capacity with CBP or SP1 was determined by measuring the CBP or SP1 immunoblot (IB) band intensity in the IP samples. The band intensity in control vector group was arbitrarily set as “1.0”. Overexpression of ERK3 ( A ) enhanced the interactions of SRC-3 with CBP and SP1 shown in ( B ). (C ) and ( D ). Cells were transfected with ERK3 siRNA (ERK3si) or the silencer negative control siRNA (Ctrlsi), together with SRC-3Flag plasmid. The interactions of SRC-3 with CBP and SP1 were analyzed by IP using a Flag Ab, followed by Western blotting. Numbers below the immunoblots of CBP and SP1 in Flag Ab/IP samples represent the relative SRC-3 binding capacity with these proteins, which is determined by the ratio of the band intensity in Flag Ab/IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity (the ratio) in Ctrlsi group was arbitrarily set as “1.0”. Knockdown of ERK3 ( C ) greatly decreased the association of SRC3 with CBP and SP1 ( D ).

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Over Expression, Negative Control

    13) Product Images from "Coordinated silencing of the Sp1-mediated long noncoding RNA MEG3 by EZH2 and HDAC3 as a prognostic factor in pancreatic ductal adenocarcinoma"

    Article Title: Coordinated silencing of the Sp1-mediated long noncoding RNA MEG3 by EZH2 and HDAC3 as a prognostic factor in pancreatic ductal adenocarcinoma

    Journal: Cancer Biology & Medicine

    doi: 10.20892/j.issn.2095-3941.2019.0427

    Schematic illustration of the coordinated silencing of the Sp1-mediated long noncoding RNA MEG3 by EZH2 and HDAC3 in pancreatic ductal adenocarcinoma. (A) Normal MEG3 regulation in normal pancreas. (B) Sp1 recruits EZH2 and HDAC3 to the MEG3 promoter and transcriptionally represses its expression. MEG3 directly sponges miR-374a-5p to regulate PTEN expression in PDAC.
    Figure Legend Snippet: Schematic illustration of the coordinated silencing of the Sp1-mediated long noncoding RNA MEG3 by EZH2 and HDAC3 in pancreatic ductal adenocarcinoma. (A) Normal MEG3 regulation in normal pancreas. (B) Sp1 recruits EZH2 and HDAC3 to the MEG3 promoter and transcriptionally represses its expression. MEG3 directly sponges miR-374a-5p to regulate PTEN expression in PDAC.

    Techniques Used: Expressing

    Suppression of Sp1, EZH2, or HDAC3 levels led to reduced MEG3 expression. (A) The promoter region of MEG3 was rich in multiple CpG islands. (B) Expression of methylated and unmethylated MEG3 in pancreatic ductal adenocarcinoma cells treated with 5-AZA-CdR, SAHA or their combination. (C) MEG3 expression in EHZ2-nc and EZH2-siRNA cells. (D) MEG3 expression in DNMT1-nc and DNMT1-siRNA cells. (E) MEG3 expression in HDAC3-nc and HDAC3-siRNA cells. (F) MEG3 expression in Sp1-nc and Sp1-siRNA cells. * P
    Figure Legend Snippet: Suppression of Sp1, EZH2, or HDAC3 levels led to reduced MEG3 expression. (A) The promoter region of MEG3 was rich in multiple CpG islands. (B) Expression of methylated and unmethylated MEG3 in pancreatic ductal adenocarcinoma cells treated with 5-AZA-CdR, SAHA or their combination. (C) MEG3 expression in EHZ2-nc and EZH2-siRNA cells. (D) MEG3 expression in DNMT1-nc and DNMT1-siRNA cells. (E) MEG3 expression in HDAC3-nc and HDAC3-siRNA cells. (F) MEG3 expression in Sp1-nc and Sp1-siRNA cells. * P

    Techniques Used: Expressing, Methylation

    Sp1, EZH2, and HDAC3 are tethered to MEG3 promoter regions as a corepressor complex to inhibit MEG3 expression. (A) The 293T cells were transfected with the HA-HDAC3 plasmid. Whole cell lysates were immunoprecipitated using antibodies against EZH2, HA, and control IgG, followed by Western blot analysis with antibodies against HA, Sp1, EZH2, and HDAC3. (B) Cell lysates were immunoprecipitated with antibodies against Sp1 and control IgG, followed by Western blot analysis with antibodies against EZH2 and HDAC3. (C) Cell lysates were immunoprecipitated with antibodies against HDAC3 and control IgG, followed by Western blot analysis with antibodies against EZH2 and Sp1. (D) ChIP assay showing Sp1 and EZH2 enrichment on MEG3 promoters. (E) Segmental chromatin immunoprecipitation assay showing Sp1 and EZH2 enrichment on MEG3 promoters in each 500 bp region. (F) Mutated locations of the MEG3 promoter (depletion mutation). (G) Luciferase activities showing that expression of wild-type MEG3 promoter was repressed by Sp1 overexpression in the -2,000~-500 bp region but not in the -500–0 bp region.
    Figure Legend Snippet: Sp1, EZH2, and HDAC3 are tethered to MEG3 promoter regions as a corepressor complex to inhibit MEG3 expression. (A) The 293T cells were transfected with the HA-HDAC3 plasmid. Whole cell lysates were immunoprecipitated using antibodies against EZH2, HA, and control IgG, followed by Western blot analysis with antibodies against HA, Sp1, EZH2, and HDAC3. (B) Cell lysates were immunoprecipitated with antibodies against Sp1 and control IgG, followed by Western blot analysis with antibodies against EZH2 and HDAC3. (C) Cell lysates were immunoprecipitated with antibodies against HDAC3 and control IgG, followed by Western blot analysis with antibodies against EZH2 and Sp1. (D) ChIP assay showing Sp1 and EZH2 enrichment on MEG3 promoters. (E) Segmental chromatin immunoprecipitation assay showing Sp1 and EZH2 enrichment on MEG3 promoters in each 500 bp region. (F) Mutated locations of the MEG3 promoter (depletion mutation). (G) Luciferase activities showing that expression of wild-type MEG3 promoter was repressed by Sp1 overexpression in the -2,000~-500 bp region but not in the -500–0 bp region.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Mutagenesis, Luciferase, Over Expression

    14) Product Images from "NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor"

    Article Title: NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor

    Journal: Data in Brief

    doi: 10.1016/j.dib.2015.11.011

    Putative transcription factor binding sites at P3 of the DDIAS promoter in HEK293 cells. (A) Putative transcription binding sites at P3 of the DDIAS promoter. (B–C) The effects of putative transcription factor knockdown were examined using a luciferase reporter assay (B); DDIAS mRNA and protein expression were evaluated using RT-PCR and Western blotting, respectively (C). HEK293 cells were transfected with siRNAs for SP1, cJun, and cMyc; scrambled siRNA was used as a control. All data are shown as means±standard errors of the mean (S.E.M).
    Figure Legend Snippet: Putative transcription factor binding sites at P3 of the DDIAS promoter in HEK293 cells. (A) Putative transcription binding sites at P3 of the DDIAS promoter. (B–C) The effects of putative transcription factor knockdown were examined using a luciferase reporter assay (B); DDIAS mRNA and protein expression were evaluated using RT-PCR and Western blotting, respectively (C). HEK293 cells were transfected with siRNAs for SP1, cJun, and cMyc; scrambled siRNA was used as a control. All data are shown as means±standard errors of the mean (S.E.M).

    Techniques Used: Binding Assay, Luciferase, Reporter Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    15) Product Images from "JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis"

    Article Title: JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01617-8

    Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway
    Figure Legend Snippet: Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway

    Techniques Used: Immunohistochemistry, Expressing

    JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P
    Figure Legend Snippet: JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P

    Techniques Used: Western Blot, Transfection, Tube Formation Assay

    JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P
    Figure Legend Snippet: JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P

    Techniques Used: Modification, Western Blot, Transfection

    16) Product Images from "Early Moderate Intensity Aerobic Exercise Intervention Prevents Doxorubicin-caused Cardiac Dysfunction through Inhibition of Cardiac Fibrosis and Inflammation"

    Article Title: Early Moderate Intensity Aerobic Exercise Intervention Prevents Doxorubicin-caused Cardiac Dysfunction through Inhibition of Cardiac Fibrosis and Inflammation

    Journal: Cancers

    doi: 10.3390/cancers12051102

    The effect of treadmill exercise on the DOX-driven upregulation of fibrosis factors Representatives of Western blot ( A ) and the densitometric analysis of TGF-β1 ( B ), phosphorylated ERK ( C ), Sp1 ( D ), and CTGF ( E ). The collagen type I ( F ) and α -SMA ( G ) were tested by ELISA. * p
    Figure Legend Snippet: The effect of treadmill exercise on the DOX-driven upregulation of fibrosis factors Representatives of Western blot ( A ) and the densitometric analysis of TGF-β1 ( B ), phosphorylated ERK ( C ), Sp1 ( D ), and CTGF ( E ). The collagen type I ( F ) and α -SMA ( G ) were tested by ELISA. * p

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay

    17) Product Images from "SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis"

    Article Title: SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0962-6

    The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P

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

    18) Product Images from "MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction"

    Article Title: MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction

    Journal: Journal of Virology

    doi: 10.1128/JVI.01311-16

    Sp1 facilitated PRRSV replication. (A to C) MARC-145 cells were transfected with control siRNA (SC) or si-Sp1, and 24 h later the cells were infected with PRRSV at an MOI of 1. Forty-eight hours later, the levels of intracellular PRRSV RNA were evaluated by qRT-PCR (A), while the virus titers in the supernatants were measured by TCID 50 (B). (C) The cell lysates were analyzed with an anti-N monoclonal antibody by Western blotting. (D to F) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or Sp1-Flag, and 24 h later the cells were infected with PRRSV at an MOI of 1. Forty-eight hours later, the cells were collected for the qRT-PCR (D), viral TCID 50 (E), and Western blotting (F). (G and H) MARC-145 cells were cotransfected with different concentrations of pcDNA3.1-Flag (NC) or Sp1-Flag and control inhibitor (IC) or miR-373 inhibitor, and then 24 h later the cells were infected with PRRSV at an MOI of 1. The cells were collected at 24 h p.i. for the qRT-PCR (G) and viral TCID 50 (H) tests. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P
    Figure Legend Snippet: Sp1 facilitated PRRSV replication. (A to C) MARC-145 cells were transfected with control siRNA (SC) or si-Sp1, and 24 h later the cells were infected with PRRSV at an MOI of 1. Forty-eight hours later, the levels of intracellular PRRSV RNA were evaluated by qRT-PCR (A), while the virus titers in the supernatants were measured by TCID 50 (B). (C) The cell lysates were analyzed with an anti-N monoclonal antibody by Western blotting. (D to F) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or Sp1-Flag, and 24 h later the cells were infected with PRRSV at an MOI of 1. Forty-eight hours later, the cells were collected for the qRT-PCR (D), viral TCID 50 (E), and Western blotting (F). (G and H) MARC-145 cells were cotransfected with different concentrations of pcDNA3.1-Flag (NC) or Sp1-Flag and control inhibitor (IC) or miR-373 inhibitor, and then 24 h later the cells were infected with PRRSV at an MOI of 1. The cells were collected at 24 h p.i. for the qRT-PCR (G) and viral TCID 50 (H) tests. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Techniques Used: Transfection, Infection, Quantitative RT-PCR, Western Blot

    PRRSV nsp9 and N induced miR-373 expression. (A) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or the indicated expression plasmid. Forty-eight hours later, miR-373 expression levels were detected by qRT-PCR. (B) MARC-145 cells were cotransfected with pGL-miR-373 and pcDNA3.1-Flag (NC) or the indicated expression plasmid. Forty-eight hours later, the cells were harvested for dual-luciferase assays. (C) MARC-145 cells were transfected with the indicated truncated miR-373 promoter reporter plasmid and phRL-TK, and 24 h later the cells were infected with PRRSV or transfected with the plasmid that expressed N or nsp9. Forty-eight hours later, the cells were harvested for dual-luciferase assays. MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or the plasmid that expressed N or nsp9, and 48 h later Sp1 expression levels were detected by qRT-PCR and Western blotting (D), while NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, IFNAR1, and IFNAR2 mRNA expression levels were detected by qRT-PCR (E). (F) Their protein levels were detected by Western blotting. (G and H) PAMs were transfected with pcDNA3.1-Flag (NC) or the plasmid that expressed N or nsp9, and 48 h later the NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, IFNAR1, and IFNAR2 mRNA expression levels were detected by qRT-PCR (G), and their protein levels were detected by Western blotting (H). Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P
    Figure Legend Snippet: PRRSV nsp9 and N induced miR-373 expression. (A) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or the indicated expression plasmid. Forty-eight hours later, miR-373 expression levels were detected by qRT-PCR. (B) MARC-145 cells were cotransfected with pGL-miR-373 and pcDNA3.1-Flag (NC) or the indicated expression plasmid. Forty-eight hours later, the cells were harvested for dual-luciferase assays. (C) MARC-145 cells were transfected with the indicated truncated miR-373 promoter reporter plasmid and phRL-TK, and 24 h later the cells were infected with PRRSV or transfected with the plasmid that expressed N or nsp9. Forty-eight hours later, the cells were harvested for dual-luciferase assays. MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or the plasmid that expressed N or nsp9, and 48 h later Sp1 expression levels were detected by qRT-PCR and Western blotting (D), while NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, IFNAR1, and IFNAR2 mRNA expression levels were detected by qRT-PCR (E). (F) Their protein levels were detected by Western blotting. (G and H) PAMs were transfected with pcDNA3.1-Flag (NC) or the plasmid that expressed N or nsp9, and 48 h later the NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, IFNAR1, and IFNAR2 mRNA expression levels were detected by qRT-PCR (G), and their protein levels were detected by Western blotting (H). Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Luciferase, Infection, Western Blot

    PRRSV infection upregulated miR-373 expression in MARC-145 cells. (A) MARC-145 cells were infected with PRRSV at an MOI of 1 for the indicated times, and the levels of miR-373 expression were measured by qRT-PCR. (B) MARC-145 cells were infected with PRRSV at different MOIs for 24 h, and then the levels of miR-373 expression were measured by qRT-PCR. (C) MARC-145 cells were transfected with pGL-miR-373, phRL-TK, or pGL4.17, and 24 h later the cells were infected with PRRSV at different MOIs. Forty-eight hours later, the cells were subjected to dual-luciferase assays. (D) 293T cells were cotransfected with different truncated miR-373 promoter report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (E) miR-373 promoter sequence of Macaca mulatta and human harbored one conserved putative GR binding site and three highly conserved putative Sp1 binding site. 293T cells were cotransfected with the indicated report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (Left) Schematic representation of mutation constructs of the Macaca mulatta miR-373 promoter. (Right) Results of dual-luciferase assays. (F) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), Sp1-Flag (800 ng), siRNA control (SC), different concentrations of si-Sp1, or different doses of Mith, and 48 h later the miR-373 promoter activity was analyzed by dual-luciferase assays and the expression levels of pri-miR-373 and miR-373 were detected by qRT-PCR. Additionally, the expression levels of Sp1 were detected by qRT-PCR and Western blotting of the corresponding group. (G) EMSA was performed as described in Materials and Methods. Biotin-labeled 40-bp probes, which included the putative Sp1 binding site (underlined) of the Macaca mulatta miRNA-373 promoter, were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled wild-type or mutant probes with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (H) ChIP assays in MARC-145 cells were performed with anti-Sp1 antibody or anti-IgG isotype control antibody. The input DNA and immunoprecipitated DNA then were purified and analyzed by qRT-PCR and PCR using primers specific for the miR-373 promoter. (I) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected for 24 h, and the expression levels of Sp1 were determined by qRT-PCR and Western blotting. (J) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later, miR-373 promoter activity was analyzed by dual-luciferase assays. (K and L) MARC-145 cells were transfected with pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later the expression levels of pri-miR-373 (K) and miR-373 (L) were detected by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P
    Figure Legend Snippet: PRRSV infection upregulated miR-373 expression in MARC-145 cells. (A) MARC-145 cells were infected with PRRSV at an MOI of 1 for the indicated times, and the levels of miR-373 expression were measured by qRT-PCR. (B) MARC-145 cells were infected with PRRSV at different MOIs for 24 h, and then the levels of miR-373 expression were measured by qRT-PCR. (C) MARC-145 cells were transfected with pGL-miR-373, phRL-TK, or pGL4.17, and 24 h later the cells were infected with PRRSV at different MOIs. Forty-eight hours later, the cells were subjected to dual-luciferase assays. (D) 293T cells were cotransfected with different truncated miR-373 promoter report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (E) miR-373 promoter sequence of Macaca mulatta and human harbored one conserved putative GR binding site and three highly conserved putative Sp1 binding site. 293T cells were cotransfected with the indicated report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (Left) Schematic representation of mutation constructs of the Macaca mulatta miR-373 promoter. (Right) Results of dual-luciferase assays. (F) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), Sp1-Flag (800 ng), siRNA control (SC), different concentrations of si-Sp1, or different doses of Mith, and 48 h later the miR-373 promoter activity was analyzed by dual-luciferase assays and the expression levels of pri-miR-373 and miR-373 were detected by qRT-PCR. Additionally, the expression levels of Sp1 were detected by qRT-PCR and Western blotting of the corresponding group. (G) EMSA was performed as described in Materials and Methods. Biotin-labeled 40-bp probes, which included the putative Sp1 binding site (underlined) of the Macaca mulatta miRNA-373 promoter, were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled wild-type or mutant probes with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (H) ChIP assays in MARC-145 cells were performed with anti-Sp1 antibody or anti-IgG isotype control antibody. The input DNA and immunoprecipitated DNA then were purified and analyzed by qRT-PCR and PCR using primers specific for the miR-373 promoter. (I) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected for 24 h, and the expression levels of Sp1 were determined by qRT-PCR and Western blotting. (J) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later, miR-373 promoter activity was analyzed by dual-luciferase assays. (K and L) MARC-145 cells were transfected with pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later the expression levels of pri-miR-373 (K) and miR-373 (L) were detected by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Sequencing, Binding Assay, Mutagenesis, Construct, Activity Assay, Western Blot, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Polymerase Chain Reaction

    Model for the mechanisms by which PRRSV infection upregulated miR-373 expression and miR-373 facilitated PRRSV replication. PRRSV, nsp9, and N upregulated miR-373 expression, since they elevated the expression of Sp1, which was an important transcriptional factor for miR-373 expression. miR-373 enhanced PRRSV replication by its negative regulation of type I interferon induction and the type I interferon receptor signaling pathway, since miR-373 targeted NFIA, NFIB, IRAK1, IRAK4, IRF1, IFR9, IFNAR1, and IFNAR2.
    Figure Legend Snippet: Model for the mechanisms by which PRRSV infection upregulated miR-373 expression and miR-373 facilitated PRRSV replication. PRRSV, nsp9, and N upregulated miR-373 expression, since they elevated the expression of Sp1, which was an important transcriptional factor for miR-373 expression. miR-373 enhanced PRRSV replication by its negative regulation of type I interferon induction and the type I interferon receptor signaling pathway, since miR-373 targeted NFIA, NFIB, IRAK1, IRAK4, IRF1, IFR9, IFNAR1, and IFNAR2.

    Techniques Used: Infection, Expressing

    19) Product Images from "TGFβ Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1"

    Article Title: TGFβ Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0363-14.2014

    TGFβ signaling modulates c-myc and p21 expression in OP cells through the cooperation of SMAD3/SMAD4 with FoxO1 and Sp1. A , FACS-sorted OPs ( CNP -EGFP low ) were cultured in the presence of TGFβ1 or Vehicle (VEH, control). B , Real-time PCR analysis for the indicated genes from cultured OPs (CNP-EGFP low ) stimulated for 8 h with TGFβ1. C , Luciferase reporter assay activity for the indicated genes was performed in CNP -EGFP low FACS-sorted cells. D–F , Immunofluorescence analysis of OP cultures after 3 h of TGFβ1 treatment shows that TGFβ signaling activation in OPs induces high levels and nuclear localization of pSMAD2/3 ( D , F ), SMAD4, FoxO1 ( E ), and Sp1 ( F ). G , H , Coimmunoprecipitation from OP cell cultures treated with TGFβ1 or vehicle for the indicated antibodies. I , J , ChIP analysis after 4 h of TGFβ1 stimulation of OP cultures. I , Diagram representing the putative binding sites of all TFs analyzed to the promoter region of p21 and c-myc genes. Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4, anti-Sp1, and anti-FoxO1 antibodies or a nonspecific rabbit IgG as a control. J , qPCR analysis revealed increased binding of these four TFs to the promoter region of c-myc and p21 , in TGFβ1-stimulated cells. Results are expressed as arbitrary units after normalization. Data are shown as mean ± SEM; n = 5 brains for each time point. * p
    Figure Legend Snippet: TGFβ signaling modulates c-myc and p21 expression in OP cells through the cooperation of SMAD3/SMAD4 with FoxO1 and Sp1. A , FACS-sorted OPs ( CNP -EGFP low ) were cultured in the presence of TGFβ1 or Vehicle (VEH, control). B , Real-time PCR analysis for the indicated genes from cultured OPs (CNP-EGFP low ) stimulated for 8 h with TGFβ1. C , Luciferase reporter assay activity for the indicated genes was performed in CNP -EGFP low FACS-sorted cells. D–F , Immunofluorescence analysis of OP cultures after 3 h of TGFβ1 treatment shows that TGFβ signaling activation in OPs induces high levels and nuclear localization of pSMAD2/3 ( D , F ), SMAD4, FoxO1 ( E ), and Sp1 ( F ). G , H , Coimmunoprecipitation from OP cell cultures treated with TGFβ1 or vehicle for the indicated antibodies. I , J , ChIP analysis after 4 h of TGFβ1 stimulation of OP cultures. I , Diagram representing the putative binding sites of all TFs analyzed to the promoter region of p21 and c-myc genes. Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4, anti-Sp1, and anti-FoxO1 antibodies or a nonspecific rabbit IgG as a control. J , qPCR analysis revealed increased binding of these four TFs to the promoter region of c-myc and p21 , in TGFβ1-stimulated cells. Results are expressed as arbitrary units after normalization. Data are shown as mean ± SEM; n = 5 brains for each time point. * p

    Techniques Used: Expressing, FACS, Cell Culture, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay, Immunofluorescence, Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

    TGFβ signaling modulates c-myc and p21 expression in OP cells during SCWM. A , G , I , Time line representing the paradigm used for TGFβ1 or vehicle treatment in vivo . B , Immunofluorescence analysis in the SCWM was performed after 4 h of TGFβ1 administration. Activation of TGFβ signaling induces nuclear localization of pSMAD2/3 ( B , C ), SMAD4 ( C , D ), and FoxO1 ( D ) in OPs. E , F , Coimmunoprecipitation analysis of SCWM extracts from TGFβ1 or vehicle (VEH)-treated mice, at 4 h after treatment. TGFβ signaling activation increased the interaction of SMAD3 and SMAD4 with FoxO1 and Sp1 in OPs in the SCWM. SCWM immune complexes were incubated with antibodies against SMAD4 ( E ) or FoxO1 ( F ). As a loading control, coimmunoprecipitation samples were blotted with the same antibodies used to perform the immunoprecipitation. G , H , P5 CNP -EGFP pups received a single administration of Veh or TGFβ1 (100 ng/kg) and 8 h later, CNP -EGFP low (OPs) were FACS sorted to isolate RNA and perform RT-PCR analysis for genes involved in the TGFβ-mediated anti-mitotic program and OL differentiation. I , J , Proteins involved in the TGFβ-mediated anti-mitotic program and OL differentiation were analyzed by WB analysis from total extracts of SCWM after 16 h of treatment. K–M , P5 CNP -EGFP mice received TGFβ1 or vehicle, and immunohistochemistry analysis was performed in the SCWM after 16 h of treatment. Representative confocal images of proteins involved in the cell cycle in OPs ( CNP -EGFP + NG2 + cells), including c-myc ( K ), p21 ( L ), and p27 ( M ) after 16 h of TGFβ1 treatment. Histograms express results in a.u. after normalization. n = 5 brains for each time point. ** p
    Figure Legend Snippet: TGFβ signaling modulates c-myc and p21 expression in OP cells during SCWM. A , G , I , Time line representing the paradigm used for TGFβ1 or vehicle treatment in vivo . B , Immunofluorescence analysis in the SCWM was performed after 4 h of TGFβ1 administration. Activation of TGFβ signaling induces nuclear localization of pSMAD2/3 ( B , C ), SMAD4 ( C , D ), and FoxO1 ( D ) in OPs. E , F , Coimmunoprecipitation analysis of SCWM extracts from TGFβ1 or vehicle (VEH)-treated mice, at 4 h after treatment. TGFβ signaling activation increased the interaction of SMAD3 and SMAD4 with FoxO1 and Sp1 in OPs in the SCWM. SCWM immune complexes were incubated with antibodies against SMAD4 ( E ) or FoxO1 ( F ). As a loading control, coimmunoprecipitation samples were blotted with the same antibodies used to perform the immunoprecipitation. G , H , P5 CNP -EGFP pups received a single administration of Veh or TGFβ1 (100 ng/kg) and 8 h later, CNP -EGFP low (OPs) were FACS sorted to isolate RNA and perform RT-PCR analysis for genes involved in the TGFβ-mediated anti-mitotic program and OL differentiation. I , J , Proteins involved in the TGFβ-mediated anti-mitotic program and OL differentiation were analyzed by WB analysis from total extracts of SCWM after 16 h of treatment. K–M , P5 CNP -EGFP mice received TGFβ1 or vehicle, and immunohistochemistry analysis was performed in the SCWM after 16 h of treatment. Representative confocal images of proteins involved in the cell cycle in OPs ( CNP -EGFP + NG2 + cells), including c-myc ( K ), p21 ( L ), and p27 ( M ) after 16 h of TGFβ1 treatment. Histograms express results in a.u. after normalization. n = 5 brains for each time point. ** p

    Techniques Used: Expressing, In Vivo, Immunofluorescence, Activation Assay, Mouse Assay, Incubation, Immunoprecipitation, FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry

    Proposed model of TGFβ signaling modulation of oligodendrogenesis during the SCWM myelination. TGFβ signaling in OPs controls the canonical downstream TGFβ-R effectors, SMAD2/3/4. Upon TGFβ-R's activation, the heteromeric SMAD2/3/4 complex establishes a nuclear localization, and cooperates with FoxO1 and Sp1 to modulate the transcription of c-myc and p21. Upon binding regulatory sequences, SMAD2/3/4, FoxO1, and Sp1 cooperate to repress the transcription of c-myc and activate p21 transcription, allowing OPs to withdraw from the cell cycle and progress down OL differentiation and maturation.
    Figure Legend Snippet: Proposed model of TGFβ signaling modulation of oligodendrogenesis during the SCWM myelination. TGFβ signaling in OPs controls the canonical downstream TGFβ-R effectors, SMAD2/3/4. Upon TGFβ-R's activation, the heteromeric SMAD2/3/4 complex establishes a nuclear localization, and cooperates with FoxO1 and Sp1 to modulate the transcription of c-myc and p21. Upon binding regulatory sequences, SMAD2/3/4, FoxO1, and Sp1 cooperate to repress the transcription of c-myc and activate p21 transcription, allowing OPs to withdraw from the cell cycle and progress down OL differentiation and maturation.

    Techniques Used: Activation Assay, Binding Assay

    20) Product Images from "PPARγ regulated CIDEA affects pro-apoptotic responses in glioblastoma"

    Article Title: PPARγ regulated CIDEA affects pro-apoptotic responses in glioblastoma

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2015.38

    Inhibition of PPAR γ reduces NF κ B and SP1 binding on CIDEA promoter. ( a ) PPAR γ inhibitior T007 has no effect on NF κ B and SP1 expression. Blot is representative of two independent experiments. Blots were re-probed for GAPDH to establish equal loading. ( b ) ChIP-qPCR assays demonstrating decreased binding of NF κ B to its cognate sites on CIDEA promoter. DNA isolated from control and PPAR γ inhibitor treated A172 glioma cells pre and post immunoprecipitation with anti-NF κ B antibody, was amplified using specific primer sets. Binding affinity of NF κ B was found to be low at three different putative binding sites on CIDEA promoter on inhibition of PPAR γ . ( c ) PPAR γ inhibition decreases SP1 binding to its cognate site on CIDEA promoter at −26 to +120 position, as indicated by ChIP-qPCR assay. Graph ( b and c ) represents fold change as calculated from Ct values of two independent experiments for a single site.
    Figure Legend Snippet: Inhibition of PPAR γ reduces NF κ B and SP1 binding on CIDEA promoter. ( a ) PPAR γ inhibitior T007 has no effect on NF κ B and SP1 expression. Blot is representative of two independent experiments. Blots were re-probed for GAPDH to establish equal loading. ( b ) ChIP-qPCR assays demonstrating decreased binding of NF κ B to its cognate sites on CIDEA promoter. DNA isolated from control and PPAR γ inhibitor treated A172 glioma cells pre and post immunoprecipitation with anti-NF κ B antibody, was amplified using specific primer sets. Binding affinity of NF κ B was found to be low at three different putative binding sites on CIDEA promoter on inhibition of PPAR γ . ( c ) PPAR γ inhibition decreases SP1 binding to its cognate site on CIDEA promoter at −26 to +120 position, as indicated by ChIP-qPCR assay. Graph ( b and c ) represents fold change as calculated from Ct values of two independent experiments for a single site.

    Techniques Used: Inhibition, Binding Assay, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Immunoprecipitation, Amplification

    21) Product Images from "TIMELESS regulates sphingolipid metabolism and tumor cell growth through Sp1/ACER2/S1P axis in ER-positive breast cancer"

    Article Title: TIMELESS regulates sphingolipid metabolism and tumor cell growth through Sp1/ACER2/S1P axis in ER-positive breast cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-03106-4

    TIM, as coactivator of Sp1, transcriptionally regulates ACER2 in breast cancer cells. a Jasper database predicts the potential Sp1 binding sequences in ACER2 promotor region. b Co-immunoprecipitation of TIM and Sp1 in MCF7 cells. c Immunocytochemistry staining of TIM and Sp1 in MCF7 and T47D cells. TIM is shown in red, Sp1 is shown in green and cell nuclei were stained with DAPI in blue. Scale bar is 10 mm. d TIM and ACER2 expression level of MCF7 and T47D cells in Sp1 knockdown and control groups. e Relative mRNA levels of TIM, Sp1 and ACER2 in MCF7 and T47D cells knockdown by siSp1. Values are means ± SD, ns: no significance; *** p
    Figure Legend Snippet: TIM, as coactivator of Sp1, transcriptionally regulates ACER2 in breast cancer cells. a Jasper database predicts the potential Sp1 binding sequences in ACER2 promotor region. b Co-immunoprecipitation of TIM and Sp1 in MCF7 cells. c Immunocytochemistry staining of TIM and Sp1 in MCF7 and T47D cells. TIM is shown in red, Sp1 is shown in green and cell nuclei were stained with DAPI in blue. Scale bar is 10 mm. d TIM and ACER2 expression level of MCF7 and T47D cells in Sp1 knockdown and control groups. e Relative mRNA levels of TIM, Sp1 and ACER2 in MCF7 and T47D cells knockdown by siSp1. Values are means ± SD, ns: no significance; *** p

    Techniques Used: Binding Assay, Immunoprecipitation, Immunocytochemistry, Staining, Expressing

    Sp1 knockdown partly rescues the effect of TIM overexpression in breast cancer cells. a Relative S1P concentration of MCF7 and T47D cells in different groups (vector+siNC, oeTIM+siNC, oeTIM+siSp1, vector+siSp1) were measured. Values are means ± SD, * p
    Figure Legend Snippet: Sp1 knockdown partly rescues the effect of TIM overexpression in breast cancer cells. a Relative S1P concentration of MCF7 and T47D cells in different groups (vector+siNC, oeTIM+siNC, oeTIM+siSp1, vector+siSp1) were measured. Values are means ± SD, * p

    Techniques Used: Over Expression, Concentration Assay, Plasmid Preparation

    22) Product Images from "Overexpression of histone deacetylases in cancer cells is controlled by interplay of transcription factors and epigenetic modulators"

    Article Title: Overexpression of histone deacetylases in cancer cells is controlled by interplay of transcription factors and epigenetic modulators

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-250654

    HDAC1 and HDAC2 promoter activity is regulated by Sp1 and Sp3 in colon cancer cell lines. A ) Analysis of HDAC1 and HDAC2 mRNA expression by qRT-PCR in HCT116, HT29, and FHs 74 Int cells. HDAC1 and HDAC2 mRNA expression is shown relative to the control
    Figure Legend Snippet: HDAC1 and HDAC2 promoter activity is regulated by Sp1 and Sp3 in colon cancer cell lines. A ) Analysis of HDAC1 and HDAC2 mRNA expression by qRT-PCR in HCT116, HT29, and FHs 74 Int cells. HDAC1 and HDAC2 mRNA expression is shown relative to the control

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR

    Sp1 or Sp3 silencing affects cell growth and colony formation in colon cancer cells. A ) Effect of Sp1 or Sp3 silencing on cell proliferation. HCT116 cells (1×10 4 ) with or without Sp1 or Sp3 KD were seeded and the cell numbers were counted every
    Figure Legend Snippet: Sp1 or Sp3 silencing affects cell growth and colony formation in colon cancer cells. A ) Effect of Sp1 or Sp3 silencing on cell proliferation. HCT116 cells (1×10 4 ) with or without Sp1 or Sp3 KD were seeded and the cell numbers were counted every

    Techniques Used:

    Effect of Sp1 on recruitment of other coregulators on HDAC1 promoter. A ) Levels of p300 or other indicated proteins in control or Sp1 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading
    Figure Legend Snippet: Effect of Sp1 on recruitment of other coregulators on HDAC1 promoter. A ) Levels of p300 or other indicated proteins in control or Sp1 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading

    Techniques Used: Western Blot, Negative Control, shRNA

    Sp1 and Sp3 affect HDAC1 and HDAC2 expression in colorectal cancer cell lines. A ) Levels of HDAC1 and other indicated proteins in control and Sp1 or Sp3 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin
    Figure Legend Snippet: Sp1 and Sp3 affect HDAC1 and HDAC2 expression in colorectal cancer cell lines. A ) Levels of HDAC1 and other indicated proteins in control and Sp1 or Sp3 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin

    Techniques Used: Expressing, Western Blot, Negative Control, shRNA

    Sp1 and Sp3 are overexpressed and bind to HDAC1 and HDAC2 promoters in colon cancer. A ) Levels of Sp1 and Sp3 proteins from various colon cancer cell lines were determined by Western blotting. Vaco250 and Vaco330 are human adenoma cell lines and used
    Figure Legend Snippet: Sp1 and Sp3 are overexpressed and bind to HDAC1 and HDAC2 promoters in colon cancer. A ) Levels of Sp1 and Sp3 proteins from various colon cancer cell lines were determined by Western blotting. Vaco250 and Vaco330 are human adenoma cell lines and used

    Techniques Used: Western Blot

    Effect of p300 KD on epigenetic markers and Sp1 binding on HDAC1 promoter. A ) Levels of HDAC1 and other indicated proteins in control or p300 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as
    Figure Legend Snippet: Effect of p300 KD on epigenetic markers and Sp1 binding on HDAC1 promoter. A ) Levels of HDAC1 and other indicated proteins in control or p300 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as

    Techniques Used: Binding Assay, Western Blot, Negative Control, shRNA

    Effect of Sp1 on recruitment of other regulators on the HDAC2 promoter. ChIP assays were carried out in HCT116 cells with Sp1 KD by using indicated antibodies: ac-H3 ( A ), H3K4 trimethylation ( B ), p300 ( C ), and SET1 ( D ). The resulting DNA was subjected
    Figure Legend Snippet: Effect of Sp1 on recruitment of other regulators on the HDAC2 promoter. ChIP assays were carried out in HCT116 cells with Sp1 KD by using indicated antibodies: ac-H3 ( A ), H3K4 trimethylation ( B ), p300 ( C ), and SET1 ( D ). The resulting DNA was subjected

    Techniques Used: Chromatin Immunoprecipitation

    23) Product Images from "Matrix metalloproteinase MMP9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer"

    Article Title: Matrix metalloproteinase MMP9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21841

    MMP9 activates EGFR1 signaling The regulation of MUC2 was investigated by using CaCo2BBE cells overexpressing MMP9 and performing WBs (25μg/lane) using whole cell lysates probed with (A) anti-EGFR, (B) anti-Sp1 and (C) anti-STAT3. The loading control for the each blot were GAPDH or β-actin. Each blot was a representation of three individual experiments. Densitometry evaluations of the WB is represented by the adjacent bar graph with each bar represents mean ± S.E., *p
    Figure Legend Snippet: MMP9 activates EGFR1 signaling The regulation of MUC2 was investigated by using CaCo2BBE cells overexpressing MMP9 and performing WBs (25μg/lane) using whole cell lysates probed with (A) anti-EGFR, (B) anti-Sp1 and (C) anti-STAT3. The loading control for the each blot were GAPDH or β-actin. Each blot was a representation of three individual experiments. Densitometry evaluations of the WB is represented by the adjacent bar graph with each bar represents mean ± S.E., *p

    Techniques Used: Western Blot

    24) Product Images from "miR-506 attenuates methylation of lncRNA MEG3 to inhibit migration and invasion of breast cancer cell lines via targeting SP1 and SP3"

    Article Title: miR-506 attenuates methylation of lncRNA MEG3 to inhibit migration and invasion of breast cancer cell lines via targeting SP1 and SP3

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0642-8

    miR-506 overexpression downregulates SP1, SP3, DNMT1 and upregulates MEG3 expression. a RT-qPCR analysis showing the level of miR-506 in control (wild-type), mimic NC- or miR-506 mimic transfected MCF-7 cells. b Western blot analysis showing the protein level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. β-actin acts as internal control. c RT-qPCR analysis showing the mRNA level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. d Bioinformatic prediction of binding site at 3′-UTR of SP1 by miR-506. e Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP1, not mutant 3′-UTR of SP1. The relative luciferase activity was measured and the data were presented as mean ± SD. f Bioinformatic prediction of binding site at 3′-UTR of SP3 by miR-506. g Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP3, not mutant 3′-UTR of SP3. The relative luciferase activity was measured and the data were presented as mean ± SD. h RT-qPCR analysis showing the level of MEG3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. i Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as negative control. j Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MDA-MB-231 cells. GAPDH acts as negative control. All data were presented as mean ± SD from three biological replicates (*P
    Figure Legend Snippet: miR-506 overexpression downregulates SP1, SP3, DNMT1 and upregulates MEG3 expression. a RT-qPCR analysis showing the level of miR-506 in control (wild-type), mimic NC- or miR-506 mimic transfected MCF-7 cells. b Western blot analysis showing the protein level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. β-actin acts as internal control. c RT-qPCR analysis showing the mRNA level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. d Bioinformatic prediction of binding site at 3′-UTR of SP1 by miR-506. e Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP1, not mutant 3′-UTR of SP1. The relative luciferase activity was measured and the data were presented as mean ± SD. f Bioinformatic prediction of binding site at 3′-UTR of SP3 by miR-506. g Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP3, not mutant 3′-UTR of SP3. The relative luciferase activity was measured and the data were presented as mean ± SD. h RT-qPCR analysis showing the level of MEG3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. i Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as negative control. j Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MDA-MB-231 cells. GAPDH acts as negative control. All data were presented as mean ± SD from three biological replicates (*P

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Activity Assay, Methylation, Polymerase Chain Reaction, Negative Control, Multiple Displacement Amplification

    25) Product Images from "SP1 and RARα regulate AGAP2 expression in cancer"

    Article Title: SP1 and RARα regulate AGAP2 expression in cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36888-x

    SP1 role in AGAP2 expression. The −246 to + 36 region of AGAP2 was analysed for SP1 binding sites with the online JASPAR prediction tool. ( a ) Representation of the SP1 binding sites location within the AGAP2 −246/+36 DNA fragment. The actual logo used for the searches is shown in ( b ) and the scores for binding are provided as a table in ( c ). ( d ) Effect of mithramycin (MTR) treatment on luciferase activity. KU812 and DU145 were transfected with the AGAP2 −246/+36 luc and the β-galactosidase plasmids and the reporter activity was measured after 24 h in the presence of 200 nM MTR or just vehicle (Vhc). Represented in the graph are the mean ± SD of data from at least three independent experiments performed in triplicates (n = 9). Values were calculated as the ratio of the luciferase activity values over the respective β-galactosidase activity and made relative to the values obtained for Vhc treated cells (control). Treatment differences were analysed with Mann-Whitney U tests (* P
    Figure Legend Snippet: SP1 role in AGAP2 expression. The −246 to + 36 region of AGAP2 was analysed for SP1 binding sites with the online JASPAR prediction tool. ( a ) Representation of the SP1 binding sites location within the AGAP2 −246/+36 DNA fragment. The actual logo used for the searches is shown in ( b ) and the scores for binding are provided as a table in ( c ). ( d ) Effect of mithramycin (MTR) treatment on luciferase activity. KU812 and DU145 were transfected with the AGAP2 −246/+36 luc and the β-galactosidase plasmids and the reporter activity was measured after 24 h in the presence of 200 nM MTR or just vehicle (Vhc). Represented in the graph are the mean ± SD of data from at least three independent experiments performed in triplicates (n = 9). Values were calculated as the ratio of the luciferase activity values over the respective β-galactosidase activity and made relative to the values obtained for Vhc treated cells (control). Treatment differences were analysed with Mann-Whitney U tests (* P

    Techniques Used: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, MANN-WHITNEY

    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 promoter (see Supplementary Table 1 ). 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.
    Figure Legend 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 promoter (see Supplementary Table 1 ). 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.

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

    26) Product Images from "Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression, et al. Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression"

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

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14663

    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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

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

    27) Product Images from "LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma"

    Article Title: LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2020.06.009

    Activation of ERK2/Sp1 signaling and recruitment of Sp1(pThr453) to the OSMR promoter induced up-regulation of OSMR. ( A ) FR180204 (100 μmol/L) and mithramycin A (500 nmol/L) treatment effectively reduced the OSMR promoter activity in Capan2 and SW1990 cells, and ( B ) inhibited OSMR expression in LLGL1-inhibited Capan2 and SW1990 cells. ( C ) Knockdown of Sp1 by siRNAs suppressed OSMR expression and ( D ) improved gemcitabine response in LLGL1-inhibited Capan2 and SW1990 cells (2-way analysis of variance, ∗∗ P
    Figure Legend Snippet: Activation of ERK2/Sp1 signaling and recruitment of Sp1(pThr453) to the OSMR promoter induced up-regulation of OSMR. ( A ) FR180204 (100 μmol/L) and mithramycin A (500 nmol/L) treatment effectively reduced the OSMR promoter activity in Capan2 and SW1990 cells, and ( B ) inhibited OSMR expression in LLGL1-inhibited Capan2 and SW1990 cells. ( C ) Knockdown of Sp1 by siRNAs suppressed OSMR expression and ( D ) improved gemcitabine response in LLGL1-inhibited Capan2 and SW1990 cells (2-way analysis of variance, ∗∗ P

    Techniques Used: Activation Assay, Activity Assay, Expressing

    LLGL1 inactivated ERK2/Sp1 signaling to suppress OSMR expression. ( A ) Increased LLGL1 expression was observed in PANC1 cells after transduction with lenti-LLGL1. ( B ) LLGL1 overexpression sensitized PANC1 cells to gemcitabine treatment (2-way analysis of variance, ∗∗∗ P
    Figure Legend Snippet: LLGL1 inactivated ERK2/Sp1 signaling to suppress OSMR expression. ( A ) Increased LLGL1 expression was observed in PANC1 cells after transduction with lenti-LLGL1. ( B ) LLGL1 overexpression sensitized PANC1 cells to gemcitabine treatment (2-way analysis of variance, ∗∗∗ P

    Techniques Used: Expressing, Transduction, Over Expression

    28) Product Images from "SP1 and RARα regulate AGAP2 expression in cancer"

    Article Title: SP1 and RARα regulate AGAP2 expression in cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36888-x

    SP1 role in AGAP2 expression. The −246 to + 36 region of AGAP2 was analysed for SP1 binding sites with the online JASPAR prediction tool. ( a ) Representation of the SP1 binding sites location within the AGAP2 −246/+36 DNA fragment. The actual logo used for the searches is shown in ( b ) and the scores for binding are provided as a table in ( c ). ( d ) Effect of mithramycin (MTR) treatment on luciferase activity. KU812 and DU145 were transfected with the AGAP2 −246/+36 luc and the β-galactosidase plasmids and the reporter activity was measured after 24 h in the presence of 200 nM MTR or just vehicle (Vhc). Represented in the graph are the mean ± SD of data from at least three independent experiments performed in triplicates (n = 9). Values were calculated as the ratio of the luciferase activity values over the respective β-galactosidase activity and made relative to the values obtained for Vhc treated cells (control). Treatment differences were analysed with Mann-Whitney U tests (* P
    Figure Legend Snippet: SP1 role in AGAP2 expression. The −246 to + 36 region of AGAP2 was analysed for SP1 binding sites with the online JASPAR prediction tool. ( a ) Representation of the SP1 binding sites location within the AGAP2 −246/+36 DNA fragment. The actual logo used for the searches is shown in ( b ) and the scores for binding are provided as a table in ( c ). ( d ) Effect of mithramycin (MTR) treatment on luciferase activity. KU812 and DU145 were transfected with the AGAP2 −246/+36 luc and the β-galactosidase plasmids and the reporter activity was measured after 24 h in the presence of 200 nM MTR or just vehicle (Vhc). Represented in the graph are the mean ± SD of data from at least three independent experiments performed in triplicates (n = 9). Values were calculated as the ratio of the luciferase activity values over the respective β-galactosidase activity and made relative to the values obtained for Vhc treated cells (control). Treatment differences were analysed with Mann-Whitney U tests (* P

    Techniques Used: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, MANN-WHITNEY

    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.
    Figure Legend 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.

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

    29) Product Images from "Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma"

    Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.15046

    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
    Figure Legend Snippet: The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P

    Techniques Used: In Vivo, Expressing, Luciferase, Mouse Assay, Injection, Imaging, Staining, Derivative Assay, Immunofluorescence

    Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P
    Figure Legend Snippet: Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P

    Techniques Used: Inhibition, Binding Assay, In Silico, Chromatin Immunoprecipitation, SPR Assay, Co-Immunoprecipitation Assay, Expressing, Planar Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Recombinant, Migration, Cell Culture

    Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P
    Figure Legend Snippet: Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P

    Techniques Used: Binding Assay, Sequencing, Luciferase, Planar Chromatography, In Vitro, Incubation, Expressing, Plasmid Preparation, Transfection, Over Expression

    30) Product Images from "High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria"

    Article Title: High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.126

    HMGA1 is located into the cytoplasm of cancer-derived cell lines and in MEFs. ( a ) Immunoblot analysis of HMGA1 expression in total (T), nuclear (N) and cytoplasmic (C) cell extracts from several breast tumor cell lines (BT549, MDA-MB-231, MDA-MB-468, T47D, MCF7), breast cell line (HBL100) (left), lung cancer cell line (H1299), cervical cancer cell line (HeLa), mouse embryonic fibroblasts (MEFs), human normal testis and seminomas (right). ( b ) Total (T), nuclear (N) and cytoplasmic (C) cell extracts from HEK293, ND7 and GC1 cells were analyzed by western blotting for the HMGA1 protein. Sp1 and γ -tubulin were used as markers of nuclear/cytoplasmic separation as well as loading controls. HMGA1 localizes at the mitochondria in the internal compartments and on the outer membrane. ( c ) Mitochondrial fractions from control and HMGA1b -transfected HEK293 cells were enzymatically digested by Proteinase K in the presence or absence of Triton X-100 (1%) and subsequently analyzed by western blotting for the indicated proteins. ( d ) Mitochondrial fractions from MDA-MB-231 cells were treated as in c and analyzed by western blotting for the indicated proteins. Cyclooxygenase IV (CoxIV), located to the inner mitochondrial membrane, was used as loading control. Cytosolic contamination was verified by western blotting using anti- γ -Tubulin antibodies
    Figure Legend Snippet: HMGA1 is located into the cytoplasm of cancer-derived cell lines and in MEFs. ( a ) Immunoblot analysis of HMGA1 expression in total (T), nuclear (N) and cytoplasmic (C) cell extracts from several breast tumor cell lines (BT549, MDA-MB-231, MDA-MB-468, T47D, MCF7), breast cell line (HBL100) (left), lung cancer cell line (H1299), cervical cancer cell line (HeLa), mouse embryonic fibroblasts (MEFs), human normal testis and seminomas (right). ( b ) Total (T), nuclear (N) and cytoplasmic (C) cell extracts from HEK293, ND7 and GC1 cells were analyzed by western blotting for the HMGA1 protein. Sp1 and γ -tubulin were used as markers of nuclear/cytoplasmic separation as well as loading controls. HMGA1 localizes at the mitochondria in the internal compartments and on the outer membrane. ( c ) Mitochondrial fractions from control and HMGA1b -transfected HEK293 cells were enzymatically digested by Proteinase K in the presence or absence of Triton X-100 (1%) and subsequently analyzed by western blotting for the indicated proteins. ( d ) Mitochondrial fractions from MDA-MB-231 cells were treated as in c and analyzed by western blotting for the indicated proteins. Cyclooxygenase IV (CoxIV), located to the inner mitochondrial membrane, was used as loading control. Cytosolic contamination was verified by western blotting using anti- γ -Tubulin antibodies

    Techniques Used: Derivative Assay, Expressing, Multiple Displacement Amplification, Western Blot, Transfection

    HMGA1 is stably present at cytoplasmic level. ( a ) Diagram of the HA-tagged Hmga2 and wild-type and Hmga1 deletion mutants used in western blotting and immunofluorescence analysis. The AT-hook domains (+) and the acidic tail (- - - - -) are indicated. ( b ) Nuclear, cytoplasmic and total cell extracts from HEK293 cells were analyzed by western blotting for the expression of the constructs. Sp1 and γ -tubulin were used as markers of nuclear/cytoplasmic separation as well as loading controls. ( c ) Subcellular localization of HA-HMGA2 and HA-HMGA1 mutant proteins in HEK293 cells transfected with the indicated vectors. Nuclei were stained with Hoechst
    Figure Legend Snippet: HMGA1 is stably present at cytoplasmic level. ( a ) Diagram of the HA-tagged Hmga2 and wild-type and Hmga1 deletion mutants used in western blotting and immunofluorescence analysis. The AT-hook domains (+) and the acidic tail (- - - - -) are indicated. ( b ) Nuclear, cytoplasmic and total cell extracts from HEK293 cells were analyzed by western blotting for the expression of the constructs. Sp1 and γ -tubulin were used as markers of nuclear/cytoplasmic separation as well as loading controls. ( c ) Subcellular localization of HA-HMGA2 and HA-HMGA1 mutant proteins in HEK293 cells transfected with the indicated vectors. Nuclei were stained with Hoechst

    Techniques Used: Stable Transfection, Western Blot, Immunofluorescence, Expressing, Construct, Mutagenesis, Transfection, Staining

    31) Product Images from "Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression, et al. Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression"

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

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14663

    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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

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

    32) Product Images from "miR-506 attenuates methylation of lncRNA MEG3 to inhibit migration and invasion of breast cancer cell lines via targeting SP1 and SP3"

    Article Title: miR-506 attenuates methylation of lncRNA MEG3 to inhibit migration and invasion of breast cancer cell lines via targeting SP1 and SP3

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0642-8

    miR-506 overexpression downregulates SP1, SP3, DNMT1 and upregulates MEG3 expression. a RT-qPCR analysis showing the level of miR-506 in control (wild-type), mimic NC- or miR-506 mimic transfected MCF-7 cells. b Western blot analysis showing the protein level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. β-actin acts as internal control. c RT-qPCR analysis showing the mRNA level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. d Bioinformatic prediction of binding site at 3′-UTR of SP1 by miR-506. e Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP1, not mutant 3′-UTR of SP1. The relative luciferase activity was measured and the data were presented as mean ± SD. f Bioinformatic prediction of binding site at 3′-UTR of SP3 by miR-506. g Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP3, not mutant 3′-UTR of SP3. The relative luciferase activity was measured and the data were presented as mean ± SD. h RT-qPCR analysis showing the level of MEG3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. i Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as negative control. j Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MDA-MB-231 cells. GAPDH acts as negative control. All data were presented as mean ± SD from three biological replicates (*P
    Figure Legend Snippet: miR-506 overexpression downregulates SP1, SP3, DNMT1 and upregulates MEG3 expression. a RT-qPCR analysis showing the level of miR-506 in control (wild-type), mimic NC- or miR-506 mimic transfected MCF-7 cells. b Western blot analysis showing the protein level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. β-actin acts as internal control. c RT-qPCR analysis showing the mRNA level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. d Bioinformatic prediction of binding site at 3′-UTR of SP1 by miR-506. e Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP1, not mutant 3′-UTR of SP1. The relative luciferase activity was measured and the data were presented as mean ± SD. f Bioinformatic prediction of binding site at 3′-UTR of SP3 by miR-506. g Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP3, not mutant 3′-UTR of SP3. The relative luciferase activity was measured and the data were presented as mean ± SD. h RT-qPCR analysis showing the level of MEG3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. i Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as negative control. j Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MDA-MB-231 cells. GAPDH acts as negative control. All data were presented as mean ± SD from three biological replicates (*P

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Activity Assay, Methylation, Polymerase Chain Reaction, Negative Control, Multiple Displacement Amplification

    33) Product Images from "Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma"

    Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.15046

    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
    Figure Legend Snippet: The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P

    Techniques Used: In Vivo, Expressing, Luciferase, Mouse Assay, Injection, Imaging, Staining, Derivative Assay, Immunofluorescence

    Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P
    Figure Legend Snippet: Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P

    Techniques Used: Inhibition, Binding Assay, In Silico, Chromatin Immunoprecipitation, SPR Assay, Co-Immunoprecipitation Assay, Expressing, Planar Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Recombinant, Migration, Cell Culture

    Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P
    Figure Legend Snippet: Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P

    Techniques Used: Binding Assay, Sequencing, Luciferase, Planar Chromatography, In Vitro, Incubation, Expressing, Plasmid Preparation, Transfection, Over Expression

    34) Product Images from "Long noncoding RNA UCA1 induced by SP1 promotes cell proliferation via recruiting EZH2 and activating AKT pathway in gastric cancer"

    Article Title: Long noncoding RNA UCA1 induced by SP1 promotes cell proliferation via recruiting EZH2 and activating AKT pathway in gastric cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.143

    The transcription factor SP1 is involved in UCA1 upregulation. ( a ) The predicted top three positions of putative SP1-binding sites in −2.5 kb human UCA1 promoter from JASPAR database. ( b ) Upregulation of SP1 and slicing of SP1 by transfecting pcDNA-SP1 and SP1 siRNAs, respectively, which were determined by western blot in the protein expression level. ( c ) RT-PCR was used to determine the expression of UCA1 in cells transfected with pcDNA-SP1 and SP1 siRNAs, respectively. Error bars represent the mean±S.D. of triplicate experiments (* P
    Figure Legend Snippet: The transcription factor SP1 is involved in UCA1 upregulation. ( a ) The predicted top three positions of putative SP1-binding sites in −2.5 kb human UCA1 promoter from JASPAR database. ( b ) Upregulation of SP1 and slicing of SP1 by transfecting pcDNA-SP1 and SP1 siRNAs, respectively, which were determined by western blot in the protein expression level. ( c ) RT-PCR was used to determine the expression of UCA1 in cells transfected with pcDNA-SP1 and SP1 siRNAs, respectively. Error bars represent the mean±S.D. of triplicate experiments (* P

    Techniques Used: Binding Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    Schematic diagram illustrating signaling of EZH2 and its upstream activator and its downstream effectors in GC. SP1 directly binds to the core promoter of UCA1 so that it activates the expression of UCA1. UCA1 directly interacts with EZH2 and increase the expression of EZH2, which affects the activation of AKT/GSK-3B/cyclin D1 axis. EZH2 could also physically interact with the cyclin D1 promoter to promote the expression of cyclin D1. In addition, P-AKT could indirectly influence the expression of EZH2 so as to form a positive feedback loop with EZH2. With the accumulation of cyclin D1, G1/S transition was activated to promote cell proliferation and cancer progression
    Figure Legend Snippet: Schematic diagram illustrating signaling of EZH2 and its upstream activator and its downstream effectors in GC. SP1 directly binds to the core promoter of UCA1 so that it activates the expression of UCA1. UCA1 directly interacts with EZH2 and increase the expression of EZH2, which affects the activation of AKT/GSK-3B/cyclin D1 axis. EZH2 could also physically interact with the cyclin D1 promoter to promote the expression of cyclin D1. In addition, P-AKT could indirectly influence the expression of EZH2 so as to form a positive feedback loop with EZH2. With the accumulation of cyclin D1, G1/S transition was activated to promote cell proliferation and cancer progression

    Techniques Used: Expressing, Activation Assay

    35) Product Images from "Adeno-associated virus-mediated brain delivery of 5-lipoxygenase modulates the AD-like phenotype of APP mice"

    Article Title: Adeno-associated virus-mediated brain delivery of 5-lipoxygenase modulates the AD-like phenotype of APP mice

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-7-1

    AAV1/2-5LO over-expression modulates CREB levels and transcription of the γ-secretase complex in the brains of Tg2576 mice . A . Levels of total CREB and its phosphorylated form at Ser133 and Sp1 in the cortex of Tg2576 receiving AAV1/2-5LO or empty vector (CTL) assayed by western blot analyses. B . Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. C . Relative mRNA levels for PS1, Nicastrin, APH-1 and Pen-2 in the cortex of Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL), as determined by real-time quantitative RT-PCR amplification. Values represent mean ± SEM (*p
    Figure Legend Snippet: AAV1/2-5LO over-expression modulates CREB levels and transcription of the γ-secretase complex in the brains of Tg2576 mice . A . Levels of total CREB and its phosphorylated form at Ser133 and Sp1 in the cortex of Tg2576 receiving AAV1/2-5LO or empty vector (CTL) assayed by western blot analyses. B . Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. C . Relative mRNA levels for PS1, Nicastrin, APH-1 and Pen-2 in the cortex of Tg2576 mice receiving AAV1/2-5LO or empty vector (CTL), as determined by real-time quantitative RT-PCR amplification. Values represent mean ± SEM (*p

    Techniques Used: Over Expression, Mouse Assay, Plasmid Preparation, CTL Assay, Western Blot, Quantitative RT-PCR, Amplification

    36) Product Images from "JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis"

    Article Title: JP3, an antiangiogenic peptide, inhibits growth and metastasis of gastric cancer through TRIM25/SP1/MMP2 axis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01617-8

    Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway
    Figure Legend Snippet: Dysregulation of the TRIM25-SP1-MMP2 axis in GC. a-c Kaplan–Meier Overall Survival curves of human GC patients with low versus high TRIM25 ( a ), combined with TRIM25/SP1 ( b ) and combined with TRIM25/MMP2 ( c ) expressions, based on TCGA data ( http://www.oncolnc.org/ ). d-e Protein levels of TRIM25 and SP1 were detected in GC tumor tissues and normal tissues by IHC. f The correlations of the TRIM25 protein levels and SP1 protein levels were calculated ( n = 90). g-h Kaplan-Meier curves depicting OS according to the expression patterns of TRIM25 ( g ) and SP1 ( h ) in the GC cohort. P values were calculated with the log-rank test. i A working model of JP3 on inhibiting tumor angiogenesis of CC via activating TRIM25 signaling pathway

    Techniques Used: Immunohistochemistry, Expressing

    JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P
    Figure Legend Snippet: JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online ( http://genemania.org/ ). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 ( c ) and SGC7901 ( d ) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). *** P

    Techniques Used: Western Blot, Transfection, Tube Formation Assay

    JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P
    Figure Legend Snippet: JP3 triggers ubiquitination modification of SP1 at K610 in GC cells. a BGC823 cells were treated with JP3 (0 or 50 μM), and then with CHX and harvested at the indicated time points for Western blotting. b The relative intensities of the SP1 protein bands were analyzed by densitometry after normalization to GAPDH. c Ubiquitination of SP1 was induced by JP3. His-ub was transfected into SGC7901 cells for 48 h and with JP3 (0 or 50 μM) for another 24 h, followed by pre-treatment with or without MG132 (10 μM) for 6 h. d The intensities of the SP1 and MMP2 protein bands in SGC7901 cells were analyzed by densitometry after normalization to Actin. e Data from the PhosphoSitePlus ( https://www.phosphosite.org ) showed the potential sites required for ubiquitination of SP1. f BGC823 cells were transfected with Flag-SP1 (WT) or mutants, followed by exposure to CHX (100 μg/ml) for 6 h. The indicated proteins were detected by Western blotting. g-h BGC823 cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h and then JP3 (50 μM) for 24 h, followed by exposure to 100 μg/ml of CHX for 0, 3, 6, 9 h; the protein level of Flag-SP1 was determined by Western blotting, and the intensity of the SP1 protein bands were analyzed ( h ). i, j BGC823 (left) and SGC7901 (right) cells were transfected with Flag-SP1 (WT) or Flag-SP1 (K610R) for 48 h, followed by treatment with JP3 (50 μM) for 24 h, and j the intensity of the SP1 protein bands were analyzed. The data are presented as the means ± SEM, ns: no significance, ** P

    Techniques Used: Modification, Western Blot, Transfection

    37) Product Images from "IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3"

    Article Title: IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.802900

    Co-operative occupancy of IL-10 promoter by K-RTA, SP1, and SP3. A, immunopurified FLAG–K-RTA (aa 1–531), SP1-HA, and SP3-HA proteins from HEK293 cells were incubated with biotin-labeled IL-10 promoter probe (fragment from nucleotides −265 to −1) in the indicated combinations, and specific protein–DNA complexes were analyzed by EMSA. Gray arrows indicate protein–DNA complexes. Specific protein–DNA complexes are numbered as 1–4 . Two independent experiments showed similar results. B, GST-pulldown experiments. Wild-type or mutant GST-K-RTA (aa 1–531) proteins were incubated with nuclear extracts, and the co-precipitated endogenous SP1 or SP3 was subsequently detected by Western blotting ( WB ). Two independent experiments showed similar results. C, DNase I footprinting assay with the IL-10 promoter DNA probe (−206/−72) labeled with [ 32 P]dATP at the 3′ terminus. The 32 P-labeled DNA probe was incubated with either immunopurified FLAG–K-RTA (0 or 2000 ng) and increasing amounts of SP3-HA (0, 55, 110, and 220 ng) An autoradiogram produced after gel electrophoresis of DNase I-digested samples is shown. A + G is DNA marker ladder by the Maxam-Gilbert A + G reaction. Boxes around predicted SP-binding site (−177/−172) and SP3-bound footprint are indicated. D, possible models of K-RTA recruitment to the IL-10 promoter. The protein–DNA complex of K-RTA, SP1, and SP3, as shown in A , may regulate IL-10 promoter transactivation. Specific protein–DNA complexes numbered as 1–4 correspond to those in A .
    Figure Legend Snippet: Co-operative occupancy of IL-10 promoter by K-RTA, SP1, and SP3. A, immunopurified FLAG–K-RTA (aa 1–531), SP1-HA, and SP3-HA proteins from HEK293 cells were incubated with biotin-labeled IL-10 promoter probe (fragment from nucleotides −265 to −1) in the indicated combinations, and specific protein–DNA complexes were analyzed by EMSA. Gray arrows indicate protein–DNA complexes. Specific protein–DNA complexes are numbered as 1–4 . Two independent experiments showed similar results. B, GST-pulldown experiments. Wild-type or mutant GST-K-RTA (aa 1–531) proteins were incubated with nuclear extracts, and the co-precipitated endogenous SP1 or SP3 was subsequently detected by Western blotting ( WB ). Two independent experiments showed similar results. C, DNase I footprinting assay with the IL-10 promoter DNA probe (−206/−72) labeled with [ 32 P]dATP at the 3′ terminus. The 32 P-labeled DNA probe was incubated with either immunopurified FLAG–K-RTA (0 or 2000 ng) and increasing amounts of SP3-HA (0, 55, 110, and 220 ng) An autoradiogram produced after gel electrophoresis of DNase I-digested samples is shown. A + G is DNA marker ladder by the Maxam-Gilbert A + G reaction. Boxes around predicted SP-binding site (−177/−172) and SP3-bound footprint are indicated. D, possible models of K-RTA recruitment to the IL-10 promoter. The protein–DNA complex of K-RTA, SP1, and SP3, as shown in A , may regulate IL-10 promoter transactivation. Specific protein–DNA complexes numbered as 1–4 correspond to those in A .

    Techniques Used: Incubation, Labeling, Mutagenesis, Western Blot, Footprinting, Produced, Nucleic Acid Electrophoresis, Marker, Binding Assay

    38) Product Images from "Baicalin induces apoptosis in SW480 cells through downregulation of the SP1 transcription factor"

    Article Title: Baicalin induces apoptosis in SW480 cells through downregulation of the SP1 transcription factor

    Journal: Anti-Cancer Drugs

    doi: 10.1097/CAD.0000000000000708

    Baicalin inhibits SW480 cells growth and decreases the expression of sp1 in SW480 human colon cancer cells. Cells were treated with DMSO or 50–400 µg/ml baicalin for 24 and 48 h. (a) The photograph of inhibition effects of baicalin on the growth of SW480 cells. (b) Inhibition rate of baicalin, cell proliferation was measured using the CCK-8 kit, as described in ‘Materials and methods’ section. (c) Baicalin decreased the protein expression of sp1 in SW480 human colon cancer cells. Each bar represents the mean±SD of three independent experiments. P
    Figure Legend Snippet: Baicalin inhibits SW480 cells growth and decreases the expression of sp1 in SW480 human colon cancer cells. Cells were treated with DMSO or 50–400 µg/ml baicalin for 24 and 48 h. (a) The photograph of inhibition effects of baicalin on the growth of SW480 cells. (b) Inhibition rate of baicalin, cell proliferation was measured using the CCK-8 kit, as described in ‘Materials and methods’ section. (c) Baicalin decreased the protein expression of sp1 in SW480 human colon cancer cells. Each bar represents the mean±SD of three independent experiments. P

    Techniques Used: Expressing, Inhibition, CCK-8 Assay

    sp1 as a determinant transcription factor of colorectal cancer during the analysis of four datasets. (a) GSE4107 gene expression according to; (b) GSE24514 gene expression according to; (c) GSE32323 gene expression according to; (b) GSE73883 gene expression according to. P
    Figure Legend Snippet: sp1 as a determinant transcription factor of colorectal cancer during the analysis of four datasets. (a) GSE4107 gene expression according to; (b) GSE24514 gene expression according to; (c) GSE32323 gene expression according to; (b) GSE73883 gene expression according to. P

    Techniques Used: Expressing

    (a, b) sp1 inhibitor mithramycin-A inhibits SW480 cells growth and decreases the expression of sp1 , (c, d) mithramycin-A induces SW480 human colon cancer cell apoptosis. Each bar represents the mean±SD of three independent experiments. P
    Figure Legend Snippet: (a, b) sp1 inhibitor mithramycin-A inhibits SW480 cells growth and decreases the expression of sp1 , (c, d) mithramycin-A induces SW480 human colon cancer cell apoptosis. Each bar represents the mean±SD of three independent experiments. P

    Techniques Used: Expressing

    39) Product Images from "Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway"

    Article Title: Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway

    Journal: Viruses

    doi: 10.3390/v11121141

    Rep protein enhances the binding activities of p50 and Sp1 with the il10 promoter at the later phase of PCV2 infection. ( A – D ) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the binding activities of p50 and Sp1 were detected at 12 h, 24 h, and 48 h using the ChIP assay ( A , B ); the relevant statistical results of ( A , B ) are shown in line graphs ( C , D ). The data of ( A , B ) are representative of three independent experiments. The data of ( C , D ) are the means ± SD of three independent experiments. ** p
    Figure Legend Snippet: Rep protein enhances the binding activities of p50 and Sp1 with the il10 promoter at the later phase of PCV2 infection. ( A – D ) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the binding activities of p50 and Sp1 were detected at 12 h, 24 h, and 48 h using the ChIP assay ( A , B ); the relevant statistical results of ( A , B ) are shown in line graphs ( C , D ). The data of ( A , B ) are representative of three independent experiments. The data of ( C , D ) are the means ± SD of three independent experiments. ** p

    Techniques Used: Binding Assay, Infection, Chromatin Immunoprecipitation

    PCV2 Rep activates p38-MAPK signaling to promote NF-κB p50 and Sp1 binding to the il10 promoter. ( A – C ) PAMs were infected with 100 MOI rAd-Rep1 (Rep1), rAd-Rep2 (Rep2), rAd-Blank (Blank), or Mock infection, and the binding levels of transcriptional factor NF-κB p50, Sp1, and AP1 to the il10 promoter were detected by the ChIP assay. ( D , E ) PAMs were transfected with the specific siRNAs of p50, p38-MAPK, or NC, and then infected with rAd-Rep2. The binding activities of p50 and Sp1 to the il10 promoter were detected by the ChIP assay. The data are the means ± SD of three independent experiments. * p
    Figure Legend Snippet: PCV2 Rep activates p38-MAPK signaling to promote NF-κB p50 and Sp1 binding to the il10 promoter. ( A – C ) PAMs were infected with 100 MOI rAd-Rep1 (Rep1), rAd-Rep2 (Rep2), rAd-Blank (Blank), or Mock infection, and the binding levels of transcriptional factor NF-κB p50, Sp1, and AP1 to the il10 promoter were detected by the ChIP assay. ( D , E ) PAMs were transfected with the specific siRNAs of p50, p38-MAPK, or NC, and then infected with rAd-Rep2. The binding activities of p50 and Sp1 to the il10 promoter were detected by the ChIP assay. The data are the means ± SD of three independent experiments. * p

    Techniques Used: Binding Assay, Infection, Chromatin Immunoprecipitation, Transfection

    Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. ( A ) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. ( B ) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 10 6 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. ( C ) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. ( D ) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. ( E , F ) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of ( A ) are representative of three independent experiments. The data of ( B – D ) are means ± SEM of three independent experiments. The data of ( E , F ) are the means ± SD of three independent experiments. * p
    Figure Legend Snippet: Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. ( A ) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. ( B ) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 10 6 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. ( C ) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. ( D ) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. ( E , F ) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of ( A ) are representative of three independent experiments. The data of ( B – D ) are means ± SEM of three independent experiments. The data of ( E , F ) are the means ± SD of three independent experiments. * p

    Techniques Used: Binding Assay, Infection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Incubation, Chromatin Immunoprecipitation

    Related Articles

    Incubation:

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism
    Article Snippet: .. Briefly, 400 μg of nuclear protein (prepared as described above) was incubated with 5 μL of anti-SP1 antibody (# 5931, Cell Signaling), at 4 o C, for 24 hours; thus, protein A-Sepharose ® (P3391, SIGMa, Merk KGaA, Darmstadt, Germany) was added, following an additional 24-hour incubation at 4 o C. After that, the antibody/protein complexes were precipitated by centrifugation (1,000 x g, 2 min) and washed 4 times. .. The pellet was used for measurement of ESR1 protein by Western blotting as described above.

    Article Title: Nicotine stimulates PPAR?/? expression in human lung carcinoma cells through activation of PI3-K/mTOR and suppression of AP-2?
    Article Snippet: .. Blots were incubated with primary antibodies against PPARβ/δ, α7 nAChR, AP-2α, AP-2β, AP-2γ or Sp1 (1:1000) overnight at 4°C, washed, and incubated with secondary anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000 dilution, Cell Signaling) for 1 h at room temperature. .. Blots were transferred to ECL solution (Pierce, Rockford IL), exposed to X-ray film and proteins were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer.

    Binding Assay:

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism
    Article Snippet: .. Besides, reduction in the binding activity observed with addition of anti-SP1 antibody confirmed the presence of SP1 protein in the complex. .. Considering that only the ESR1-mediated regulation of Slc2a4 expression could involve the participation of SP1, EMSA analysis was performed in cells treated with E2, PPT or both.

    Activity Assay:

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism
    Article Snippet: .. Besides, reduction in the binding activity observed with addition of anti-SP1 antibody confirmed the presence of SP1 protein in the complex. .. Considering that only the ESR1-mediated regulation of Slc2a4 expression could involve the participation of SP1, EMSA analysis was performed in cells treated with E2, PPT or both.

    Centrifugation:

    Article Title: Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism
    Article Snippet: .. Briefly, 400 μg of nuclear protein (prepared as described above) was incubated with 5 μL of anti-SP1 antibody (# 5931, Cell Signaling), at 4 o C, for 24 hours; thus, protein A-Sepharose ® (P3391, SIGMa, Merk KGaA, Darmstadt, Germany) was added, following an additional 24-hour incubation at 4 o C. After that, the antibody/protein complexes were precipitated by centrifugation (1,000 x g, 2 min) and washed 4 times. .. The pellet was used for measurement of ESR1 protein by Western blotting as described above.

    Blocking Assay:

    Article Title: Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart
    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. .. Secondary antibodies conjugated to IRDye TM 800 (1:15 000, Rockland, USA) were detected using an Odyssey infrared imaging system (LI-COR, USA).

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    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: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    ESR1-mediated stimulation of Slc2a4 gene transcription involves a <t>SP1's</t> cooperative mechanism. A , . 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
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    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).
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    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

    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

    ESR1-mediated stimulation of Slc2a4 gene transcription involves a SP1's cooperative mechanism. A , . 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 , . 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: Briefly, 400 μg of nuclear protein (prepared as described above) was incubated with 5 μL of anti-SP1 antibody (# 5931, Cell Signaling), at 4 o C, for 24 hours; thus, protein A-Sepharose ® (P3391, SIGMa, Merk KGaA, Darmstadt, Germany) was added, following an additional 24-hour incubation at 4 o C. After that, the antibody/protein complexes were precipitated by centrifugation (1,000 x g, 2 min) and washed 4 times.

    Techniques: Sequencing, Binding Assay, Electron Paramagnetic Resonance, 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: Briefly, 400 μg of nuclear protein (prepared as described above) was incubated with 5 μL of anti-SP1 antibody (# 5931, Cell Signaling), at 4 o C, for 24 hours; thus, protein A-Sepharose ® (P3391, SIGMa, Merk KGaA, Darmstadt, Germany) was added, following an additional 24-hour incubation at 4 o C. After that, the antibody/protein complexes were precipitated by centrifugation (1,000 x g, 2 min) and washed 4 times.

    Techniques: Activity Assay, Expressing, Staining, Sequencing

    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