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: The effect of differentiation and TGFβ on mitochondrial respiration and mitochondrial enzyme abundance in cultured primary human skeletal muscle cells

doi: 10.1038/s41598-017-18658-3

Figure Lengend Snippet: Antibodies and primers used.

Article Snippet: MCAD , IB , 1:400 , Santa Cruz Biotech, Dallas, Texas, USA , sc365448.

Techniques: