Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 3. ������������������������������������������������������������������������������������������������������ ����������������������������������������� Flagellin-induced expression of Notch in colon epithelial cells. (A and B) Mice were anesthetized for 90-120 min with an intra‑peritoneal pento barbital injection and then given an intrarectal administration of TNBS (100 µl, 125 mg/kg) dissolved in 50% ethanol, with or without flagellin (40 µg/kg) or LPS (80 µg/kg). Colonic epithelial cells were then harvested for Notch1 and Jagged1 expression from the extracted total RNA by real‑time PCR at different time‑points. β‑actin served as the internal control intrarectal flagellin injection induces the Notch1 and Jagged1 expression in a time‑dependent manner. *p<0.05 vs. Flagellin (-) or LPS (-). Error bars indicate the SEM for values obtained from three independent experiments. (C) Colon‑26 cells (1x105 cells) were cultured in 6‑well plates in the presence or absence of flagellin at various time points, and Hes1 expression was assessed using real‑time PCR. Flagellin sig nificantly induced the Hes1 expression in Colon‑26 cells. *p<0.05 vs. Flagellin(-)/Jagged1‑Fc(-), respectively. Error bars indicate the SEM for values obtained from three independent experiments. (D) Effects of flagellin on RBP‑Jκ‑mediated reporter gene expression. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates. After 12‑16 h, the cells were transfected with an RBP‑Jκ reporter construct with an internal control vector, as described in the manufacturer's instructions. Similarly, co‑transfection with mock and DN‑TLR5 vectors was also performed using Lipofectamine 2000 in corresponding wells. At 12 h after transfection, the cells were treated with DAPT (10 µM), Jagged1-Fc (5 µg/ml) and flagellin (10 ng/ml) for 12 h. A dual luciferase assay was then performed using total cell extracts. A significant induction of N1ICD‑RBP‑Jk‑mediated reporter gene expression following flagellin stimulation was observed. RBP-Jκ activity was increased in the presence of Notch ligand Jagged1, whereas it was reduced in the presence of DAPT. **p<0.01, *p<0.05 vs. Flagellin(-) Control; #p<0.05 vs. Flagellin(+) Control. Error bars indicate the SEM for values obtained from three independent experiments. (E) In order to block the endogenous expression of Jagged1, commercially available mouse Jagged1‑specific siRNA was used. For siRNA transfection, Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates. After 12-16 h, the cells were transfected with 20 pmols each of duplex siRNAs for Jagged1 or non‑target‑specific negative control, using the siRNA‑trasfection reagent. To determine inhibition of the target gene, the Jagged1 expression was assessed by real‑time PCR at ~24‑36 h after transfection. *p<0.05 vs. negative control siRNA. Error bars indicate the SEM for values obtained from three independent experiments. (F) Effects of Jagged1 siRNA on flagellin‑induced RBP‑Jκ reporter gene expression. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates. After 12‑16 h, the cells were co‑transfected with 20 pmols each of duplex siRNAs for Jagged1 or negative control and RBP‑Jκ reporter constructs with internal control using trasfec tion reagent, as described in the manufacturer's instructions. At 24-36 h after transfection, the cells were treated with flagellin (100 ng/ml) for 12 h, after which a dual luciferase assay was carried out using total cell extracts. Knockdown of Jagged1 significantly reduced the flaggelin effects on RBP-Jκ activity. *p<0.05 vs. Flagellin(-) siRNA(-)/NC‑si; #p<0.05 vs. Flagellin(+) siRNA(-)/NC‑si. Error bars indicate the SEM for values obtained from three independent experiments.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Control, Cell Culture, Gene Expression, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Blocking Assay, Negative Control, Inhibition, Knockdown

Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 4. (A) In vitro effects of Notch on TLR5‑mediated NF‑κB signaling with or without treatment of NEMO‑binding domain (NBD) inhibitor peptide. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates for 12‑16 h, then transfected with pNF‑κB‑luc and pRL‑TK‑luc vectors using Lipofectamine 2000. At 12 h after transfection, the cells were treated with or without NBD inhibitor peptide (100 µM), DAPT (10 µM), recombinant Jagged1 (5 µg/ml) and flagellin (100 ng/ml). N.S., not statistically significant. **p<0.01, *p<0.05 vs. Flagellin(-) DMSO; #p<0.05 vs. Flagellin(+) DMSO. Error bars indicate the SEM for values obtained from three independent experiments. (B-E) In vitro effects of Notch on TLR5‑mediated IL‑6 activation. (B) Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates in the presence or absence of flagellin (10 ng/ml) at various time‑points. IL‑6, IL‑1β and TNF‑α expression was assessed by real‑time PCR from the extracted RNA samples at different time‑points. Error bars indicate the SEM for values obtained from three independent experiments. (C) Mouse IL‑6 promoter construct, pIL‑6/1278‑luc (200 ng/well) and pRL‑TK‑luc (20 ng/well) were transfected using Lipofectamine 2000 (2.5 µl/well) into Colon‑26 cells (2.5x104 cells/well), plated in 24‑well cell culture plates. Following treatment for 12 h with or without DMSO, DAPT (10 µM), Jagged1-Fc (5 µg/ml) and flagellin (10 ng/ml) for 12 h, dual luciferase assays were performed with extracted proteins. **p<0.01, *p<0.05 vs. Flagellin(-) DMSO; #p < 0.05 vs. Flagellin(+) DMSO. Error bars indicate the SEM for values obtained from three independent experiments. (D) For RBP‑Jκ‑siRNA transfection, Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates. After 12‑16 h, the cells were transfected with 20 pmols each of duplex siRNAs for RBP‑Jκ- or non‑target‑specific negative control, using the siRNA‑trasfection reagent. Inhibition of RBP‑Jκ expression was assessed by real‑time PCR at ~24‑36 h after transfection. *p<0.05 vs. negative control siRNA. Error bars indicate the SEM for values obtained from three independent experiments. (E) At 24‑36 h after siRNA tansfection, Colon‑26 cells were stimulated with flagellin (10 ng/ml) and the IL‑6 expression was assessed at different time‑points. *p<0.05 vs. Flagellin(-) siRNA(-)/NC-si; #p<0.05 vs. Flagellin(+) siRNA(-)/NC‑si. Error bars indicate the SEM for values obtained from three independent experiments.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: In Vitro, Transfection, Recombinant, Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Construct, Cell Culture, Luciferase, Negative Control, Inhibition

Journal: PloS one

Article Title: RBPJ is a novel target for rhabdomyosarcoma therapy.

doi: 10.1371/journal.pone.0039268

Figure Lengend Snippet: Figure 1. Notch pathway molecules are overexpressed in rhabdomyosarcoma cells. Notch pathway genes (receptors NOTCH1-4, ligands JAG1 and DLL1, target genes HES1 and HEY1, and transcription factor RBPJ) were assessed by real-time PCR in a normal human skeletal muscle specimen and 2 human RMS biopsy specimens. The Ct values of all RMS samples were normalized to those of ACTB. The values of the human RMS specimens were compared with those of the human skeletal muscle sample, which is defined as a relative expression of 1.0. Columns, mean values of 3 independent experiments; bar, SD. *p,0.05, **p,0.01. doi:10.1371/journal.pone.0039268.g001

Article Snippet: Plasmid Constructs and Gene Transfer Control siRNA (S20C-0600) was purchased from B-Bridge International (Cupertino, USA) and RBPJ siRNA (sc-38214) was purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: PloS one

Article Title: RBPJ is a novel target for rhabdomyosarcoma therapy.

doi: 10.1371/journal.pone.0039268

Figure Lengend Snippet: Figure 3. Knockdown of RBPJ suppresses anchorage-independent growth of rhabdomyosarcoma cells. A, The expression of RBPJ mRNA in RD cells was assessed by real-time PCR. The Ct values of all RMS cell lines were normalized to those of ACTB. The values of the RMS cell lines were compared with HSkMc cell, which is defined as a relative expression of 1.0. B, RBPJ protein levels in RD cells transfected with control and RBPJ siRNA

Article Snippet: Plasmid Constructs and Gene Transfer Control siRNA (S20C-0600) was purchased from B-Bridge International (Cupertino, USA) and RBPJ siRNA (sc-38214) was purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control

Journal: PloS one

Article Title: RBPJ is a novel target for rhabdomyosarcoma therapy.

doi: 10.1371/journal.pone.0039268

Figure Lengend Snippet: Figure 4. Overexpression of RBPJ promotes rhabdomyosarcoma cell growth. A, RBPJ protein levels in RD cells transfected with control vector and RBPJ overexpression vector were measured by Western blotting analysis (top). RBPJ and HES1 mRNA in RD cells transfected with control vector and RBPJ overexpression vector were assessed by real-time PCR analysis. Ct values of RBPJ and HES1 were normalized to ACTB. Comparison was made to the RD cells transfected control vector, which is defined as a relative expression of 1.0 (bottom). B, Anchorage-independent growth in RD cells transfected with control vector and RBPJ overexpression vector were evaluated by colony formation assay. Fourteen days later, the each colonies were counted and pictured. Scale bar is 200 mM. Columns, mean values of 3 independent experiments; bar, SD. *p,0.05, **p,0.01. doi:10.1371/journal.pone.0039268.g004

Article Snippet: Plasmid Constructs and Gene Transfer Control siRNA (S20C-0600) was purchased from B-Bridge International (Cupertino, USA) and RBPJ siRNA (sc-38214) was purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Comparison, Expressing, Colony Assay