sp3  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Medicine

Article Title: A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression

doi: 10.3892/ijmm.2017.3273

Figure Lengend Snippet: Evaluation of the binding activities of the identified proteins. (A) INS-1 cell nuclear extracts and purified fractions were subjected to immunoblotting with specific antibodies. Except for Sp3, 10% of the nuclear extract used for purification were loaded onto a gel. For Sp3, 50% of the nuclear extract was used for electrophoresis. (B) In vitro radiolabeled proteins were individually mixed with DNA-immobilized nanobeads of the single nucleotide polymorphism rs163184 region (risk or NR) or (−). Input and eluate fractions were separated using SDS-PAGE and visualized by autoradiography. Except for Sp3, 10% of the samples used for binding assay were loaded onto a gel. For Sp3, 50% of the sample was subjected to electrophoresis. NR, non-risk; (−), negative control nanobeads without immobilized DNA.

Article Snippet: Several cell lines (HeLa, MCF-7, KP-3, KP-4, 293T and Caco-2) were seeded (2×10 5 cells/well) into 24-well plates and trans-fected with either a control small interfering RNA (siRNA) (10 nM; cat. no. 4390844; Thermo Fisher Scientific, Inc.) or siRNA (10 nM) targeting human SP3 (cat. no. s13324; Silencer Select siRNA; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000.

Techniques: Binding Assay, Purification, Western Blot, Electrophoresis, In Vitro, SDS Page, Autoradiography, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression

doi: 10.3892/ijmm.2017.3273

Figure Lengend Snippet: Allele-specific binding activity of Sp3 for the genomic region containing SNP rs163184. Cells were transfected with expression vectors for Sp3-Myc-FLAG. DNA-protein complexes were then immunoprecipitated with the anti-Myc-tag antibody. Enrichment of the target genomic regions was estimated by quantitative polymerase chain reaction analysis, and the fold enrichments of the SNP rs163184 region vs. the region 3,000 base pairs upstream and the region 3,000 base pairs downstream were determined. * P<0.01 vs. the upstream group. NR, non-risk; SNP, single nucleotide polymorphism.

Article Snippet: Several cell lines (HeLa, MCF-7, KP-3, KP-4, 293T and Caco-2) were seeded (2×10 5 cells/well) into 24-well plates and trans-fected with either a control small interfering RNA (siRNA) (10 nM; cat. no. 4390844; Thermo Fisher Scientific, Inc.) or siRNA (10 nM) targeting human SP3 (cat. no. s13324; Silencer Select siRNA; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000.

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

Journal: International Journal of Molecular Medicine

Article Title: A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression

doi: 10.3892/ijmm.2017.3273

Figure Lengend Snippet: Effect of SP3 knockdown on the expression of KCNQ1 and nearby genes. Cell lines were transfected with control siRNA or siRNA that targeted human SP3 . Gene expression levels were determined by reverse transcription-quantitative polymerase chain reaction analysis and normalized to β-actin. The expression level of each gene using negative control siRNA was designated as 1.0 in each cell line. The data for TRPM5 and SLC22A18AS from all cell lines, as well as the data for KCNQ1 and OSBPL5 from Caco-2 cells, have been excluded because of the limit of detection. * P<0.01 vs. negative control siRNA samples. siRNA, small interfering RNA; NR, non-risk.

Article Snippet: Several cell lines (HeLa, MCF-7, KP-3, KP-4, 293T and Caco-2) were seeded (2×10 5 cells/well) into 24-well plates and trans-fected with either a control small interfering RNA (siRNA) (10 nM; cat. no. 4390844; Thermo Fisher Scientific, Inc.) or siRNA (10 nM) targeting human SP3 (cat. no. s13324; Silencer Select siRNA; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression

doi: 10.3892/ijmm.2017.3273

Figure Lengend Snippet: Proposed mechanisms of gene regulation mediated by single nucleotide polymorphism rs163184, Sp3 and Lsd1/Kdm1a.

Article Snippet: Several cell lines (HeLa, MCF-7, KP-3, KP-4, 293T and Caco-2) were seeded (2×10 5 cells/well) into 24-well plates and trans-fected with either a control small interfering RNA (siRNA) (10 nM; cat. no. 4390844; Thermo Fisher Scientific, Inc.) or siRNA (10 nM) targeting human SP3 (cat. no. s13324; Silencer Select siRNA; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000.

Techniques:

Journal: International journal of biological sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Figure 7. Transcriptional regulation of CPTP expression by Sp1/Sp3. (A) 5’ deletion analysis of the CPTP promoter in the PANC-1 cell line. A series of CPTP promoter deletion mutants were generated utilizing firefly luciferase plasmid. Each construct was co-transfected with pRL-TK plasmid as the internal control. Numbering is relative to the major transcriptional start site. (B) EMSA for the -282/-273 binding site. The assays were performed with nuclear extracts from the PANC-1 or Miacapa-2 cell lines. Competitive assays were executed with a 200-fold molar excess of unlabeled double-strand probes. Supershift assay was carried out with adding Sp1 or Sp3 antibody. The arrows show specific transcription factor binding. # indicates supershifted bands. #? indicates the position of expected supershifted Sp1 complex. (C) EMSA for the -258/-249 binding site. (D) Sp1/Sp3 interacts with the CPTP promoter in vivo was identified by ChIP assay, mouse IgG and the CPTP region (+67/+251) which does not contain binding sites of Sp1/Sp3 acted as negative control. Input, sheared DNA preceding immunoprecipitation posed as positive control. (E) RT-qPCR analysis for CPTP mRNA expression levels in the PANC-1 cells treated with mithramycin A for 24 h. (F) Knockdown of Sp1 or Sp3 decreased CPTP promoter activity. Cells transfected with pGL3(-454/-1), then transfected with Sp1 or Sp3 siRNA, and cultured for 24 h before dual-luciferase activity assay and Western blot analysis. *P < 0.05, **P < 0.01.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing, Generated, Luciferase, Plasmid Preparation, Construct, Transfection, Control, Binding Assay, In Vivo, Negative Control, Immunoprecipitation, Positive Control, Quantitative RT-PCR, Knockdown, Activity Assay, Cell Culture, Western Blot

Journal: International journal of biological sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Figure 8. Sp3 attenuates the decrease in cell proliferation, migration and invasion induced by CPTP knockdown. (A) Decreased cell viability in CPTP knockdown PC cells was restored by Sp3 overexpression. The cell viability was measured using a CCK-8 assay. (B-C) Sp3 overexpression rescued the decrease in cell motility in CPTP knockdown PC cells. (D) Sp3 overexpression rescued upregulation of epithelial-related markers and downregulation of mesenchymal-related markers expression induced by CPTP knockdown as detected by Western blot analysis. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Migration, Knockdown, Over Expression, CCK-8 Assay, Expressing, Western Blot

Journal: International journal of biological sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Figure 9. Sp1/Sp3 expression is concurrently increased with CPTP expression in PC tissues. Representative images and IHC scores of Sp1 (A-B) or Sp3 (C-D) expression in PC (90 cases) and adjacent normal tissues (60 cases) were detected using IHC. Scale bar, 500 μm. Correlation analysis of protein expression in PC tissue samples between CPTP and Sp1 (E) or Sp3 (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing

Journal: International journal of biological sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Figure 10. Schematic diagram illustrating the proposed mechanism of CPTP in PC cells. CPTP expression affects the levels of sphingolipid metabolite ceramide as well as the PI4KA/AKT signaling pathways. At the point when CPTP expression is upregulated, activation of the PI4KA/AKT signaling pathway and decreased levels of the sphingolipid metabolite ceramide promote PC cell growth and metastasis, respectively. Furthermore, in PC cells, the transcription factors Sp1 and Sp3 operate as upstream positive regulators of CPTP expression. Cer, ceramide.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing, Protein-Protein interactions, Activation Assay

Journal: Nutrients

Article Title: Roseburia intestinalis and Its Metabolite Butyrate Inhibit Colitis and Upregulate TLR5 through the SP3 Signaling Pathway.

doi: 10.3390/nu14153041

Figure Lengend Snippet: Figure 3. Expression levels of the flagellin recognition receptor TLR5 and its potential transcription factor in the control, DSS and DSS + R. I groups treated mice. (A) Expression levels of TLR5, Sp1, and Sp3 in the three groups as determined by RT–qPCR analysis. (B) Correlation analysis between TLR5 and Sp3 in the three groups. (C) Correlation analysis between TLR5 and Sp1 in the three groups. (D) Representative images of TLR5 and Sp3 immunohistochemical staining in colon sections from the three groups. (E) Representative Western blot analysis showing TLR5 and Sp3 in colon sections from the three groups. Control vs. DSS, Control vs. DSS + R. I, and DSS vs. DSS + R. I; * p < 0.05 and ** p < 0.01.

Article Snippet: Sp3 siRNA and control siRNA, purchased from Santa Cruz Biotechnology, were transfected into HT29 cells with riboFECT mRNA Transfection Reagent (RiboBio, Wuhan, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot

Journal: Nutrients

Article Title: Roseburia intestinalis and Its Metabolite Butyrate Inhibit Colitis and Upregulate TLR5 through the SP3 Signaling Pathway.

doi: 10.3390/nu14153041

Figure Lengend Snippet: Figure 6. Butyrate regulates TLR5 expression through the transcription factor Sp3. (A) Control siRNA and Sp3-specific siRNA with and without butyrate were transfected into HT29 cells. After 48 h, Sp3 mRNA expression and TLR5 mRNA expression (B) were detected by RT–qPCR. (C) Western blot analysis and quantification of TLR5 and Sp3 protein expression after transfection with control siRNA and Sp3-specific siRNA in the control and butyrate groups. (D,E) A representative image of Western blot analysis. (F) ChIP-sequencing analysis of Sp3 in HT29 cells treated with butyrate. ChIP assays with H3 and Sp3 antibodies in butyrate-treated or untreated HT29 cells. qPCR showed H3 and Sp3 binding to the TLR5 promoter or RPL30 promoter. * p < 0.05 and ** p < 0.01.

Article Snippet: Sp3 siRNA and control siRNA, purchased from Santa Cruz Biotechnology, were transfected into HT29 cells with riboFECT mRNA Transfection Reagent (RiboBio, Wuhan, China).

Techniques: Expressing, Control, Transfection, Quantitative RT-PCR, Western Blot, ChIP-sequencing, Binding Assay

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Prediction of transcription factor binding sites and mutation analysis of the mSTING minimal promoter. ( A ) A schematic representation of the putative binding sites for DNA-binding proteins in the proximal promoter of the mSTING gene. The 129 nt DNA sequence (from −77 to +52) was analyzed with TFSEARCH softwares. The putative transcription factor binding sites were outlined with black box, and the names of the transcription were annotated. The transcription start site (TSS) was marked by black arrow. ( B ) Conserved base sequence of GATA1, IK2, Sp1/Sp3 or STAT binding site. The base size presented the binding affinity coefficient of transcription factor and promoter. ( C ) Mutation analysis of the mSTING minimal promoter. The left: Binding sites for GATA1, IK2, Sp1/Sp3 and STAT were indicated with open different shapes. Mutations were shown in bold above the histogram. The right: The relative luciferase activities derived from mutational pSTING-254. The site-special mutagenized plasmids were cotransfected with pRL-TK into NIH3T3 cells and lucifarase assays were performed. The level of firefly luciferase activities was normalized to the Renilla luciferase activity. Each bar represented the mean ± SD of three independent experiments. (n = 3, * p < 0.05 vs. pSTING-254).

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Binding Assay, Mutagenesis, DNA Binding Assay, Sequencing, Luciferase, Derivative Assay, Activity Assay

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Effect of GATA1 on promoter activity of the mSTING gene. ( A ) Expression of the exogenous GATA1 increased the luciferase reporter gene activity of STING and knockdown of the endogenous GATA1 decreased the promoter activity. NIH3T3 cells were cotransfected with plasmids GATA1 or pcDNA 3.1(+) (200 ng), GATA1 siRNA or negative control siRNA (50 nM) and pSTING-254 (100 ng), pRL-TK (3 ng). Luciferase assays were performed after 24 h. ( B – D ) The mRNA and protein levels of mSTING were significantly increased after GATA1 overexpression. qRT-PCR and western blot were performed after GATA1 plasmid or pcDNA 3.1(+) were transiently cotransfected into NIH3T3 cells. The protein expression was quantified to GAPDH. Each bar represented the mean ± SD of three independent experiments (n = 3, * P < 0.05 vs. Control). ( E – G ) Knockdown of GATA1 by siRNA (50 nM) reduced the mRNA and protein level of mSTING. The protein expression was quantified to GAPDH. siRNA negative control was set as 1. Each bar represented the mean ± SD of three independent experiments (n = 3, * P < 0.05 vs. NC).

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Activity Assay, Expressing, Luciferase, Knockdown, Negative Control, Over Expression, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Sp3 but not Sp1 regulated the mSTING proximal promoter activity. ( A , B ) Overexpression of Sp1 or knockdown of Sp1 had no effect on the activity of the mSTING promoter. While knockdown of Sp3 by siRNA reduced the mSTING promoter activity to 35%, and overexpression of Sp3 increased the promoter activity by more than 2 folds. NIH3T3 cells were co-transfected with Sp1/Sp3 siRNA (50 nM) or Sp1/Sp3 expressing plasmid (200 ng) and pSTING-254(100 ng) and inner control pRL-TK (3 ng). After 24 h, luciferase activity was measured. ( C , E , G ) Reduction of endogenous Sp3 expression by siRNA decreased expression of the mSTING gene at mRNA and protein level. qRT-PCR and western blot were performed after siRNA/NC(50 nM) were transiently cotransfected into NIH3T3 cells. siRNA negative control was set as 1. Each bar represented the mean ± SD of three independent experiments. (n = 3, * P < 0.05 vs.NC). ( D , F , H ) STING protein expression increased by two times under overexpression of Sp3. Sp3 plasmid, pcDNA 3.1(+) (4000 ng) were transiently cotransfected into NIH3T3 cells, and then qRT-PCR and western blot were performed. Each bar represented the mean ± SD of three independent experiments. (n = 3, * P < 0.05 vs. Control).

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Activity Assay, Over Expression, Knockdown, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: ChIP assay of the mSTING proximal promoter was performed in NIH3T3 cells. The immunoprecipitated chromatin fragments were analyzed by semi-quantitative PCR using primer pairs spanning the putative GATA1 and Sp1 binding site (the target locus) or exon 6 of the mSTING (a non-target locus). ( A ) GATA1 bound to the mSTING promoter in vivo . A band of 199 nt containing the GATA1 binding site in the mSTING promoter region was amplified. Lane M: DNA marker 1000. Lane INPUT: PCR product derived by ChIP-GATA1 primers from direct input DNA template without immunoprecipitation. Lane IgG: PCR product derived by ChIP-GATA1 primers from DNA template immunoprecipitated by normal IgG as a negative control. Lane GATA1: PCR product derived by ChIP-GATA1 primers from DNA template immunoprecipitated by anti-GATA1 antibody. Lane STING: PCR product derived by primers flanking exon 6 of the mSTING from DNA template immunoprecipitated by anti-GATA1 antibody. ( B ) Sp3 bound to the mSTING promoter in vivo . A band of 107 nt containing Sp3 binding site in the mSTING promoter region was amplified. Lane M: DNA marker 1000. Lane INPUT: PCR product derived by ChIP-Sp3 primers from direct input DNA template without immunoprecipitation. Lane IgG: PCR product derived by ChIP-Sp3 primers from DNA template immunoprecipitated by normal IgG as a negative control. Lane Sp3: PCR product derived by ChIP-Sp3 primers from DNA template immunoprecipitated by anti-Sp3 antibody. Lane STING: PCR product derived by primers flanking exon 6 of them STING from DNA template immunoprecipitated by anti-Sp3 antibody.

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, In Vivo, Amplification, Marker, Derivative Assay, Negative Control

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Sequences of oligonucleotides used in RT-PCR and ChIP assay.

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: