Journal: PLoS ONE

Article Title: Activated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways

doi: 10.1371/journal.pone.0020537

Figure Lengend Snippet: A, Left panel, Re-expression of miR-200b was established in Rink-1 cells by transfection with its precursor. Middle panel, Re-expression of miR-200b did not regulate the expression of Notch receptors in Rink-1 cells. Right panel, Re-expression of miR-200b regulated the expression of Jagged-1 and Jagged-2 mRNAs in Rink-1 cells. B, Left and middle panel, Re-expression of miR-200b inhibited the expression of Jagged-1 target genes at mRNA and protein levels in Rink-1 cells. Right panel, Re-expression of miR-200b inhibited Rink-1 cell growth test by MTT assay. C, Left and middle panel, Jagged-1 siRNA inhibited the expression of Jagged-1 target gene Hes-1 and Hey-1 at mRNA and protein levels in Rink-1 cells. Right panel, Jagged-1 siRNA inhibited Rink-1 cell growth test by MTT assay.

Article Snippet: Cells were transfected with 100 nmol/L of Notch-1, Notch-2, Notch-3, Notch-4, Jagged-1 siRNA or control siRNA (Santa Cruz) as well as 20 nmol/L of miR-200b (Ambion, Austin, TX) using DharmaFECT3 siRNA transfection reagent (DHARMACON, Lafayette, CO) as previously described .

Techniques: Expressing, Transfection, MTT Assay

Journal: PLoS ONE

Article Title: Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

doi: 10.1371/journal.pone.0063896

Figure Lengend Snippet: A) RT-PCR of Notch-1 expression. B,C) Western blotting analysis of activated intracellular form of Notch-1 (Notch-ICD, B) ) and of Notch-1 ligand, Jagged-1(C). The densitometric analyses of the bands normalized to GAPDH are reported in the histograms. D) Confocal immunofluorescence analysis of Notch-1 (green) and Hes-1 (cyan) expression. For the analysis of Notch-1, the cells were stained with a specific antibody recognizing both the membrane Notch-1 receptor and its activated intracellular form, Notch-ICD. Densitometric analyses of Notch-ICD and Hes-1 fluorescent signals are reported in the corresponding histogram. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β1.

Article Snippet: To inhibit the expression of Jagged-1, short interference RNA duplexes (siRNA) (Santa Cruz) were used essentially as describred previously .

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

doi: 10.1371/journal.pone.0063896

Figure Lengend Snippet: Neonatal cardiac fibroblasts were cultured for 48 h and treated as indicated. A) Western blotting analysis of NICD expression in the absence (control) or presence of DAPT (5 µM) a pharmacological γ-secretase inhibitor, used to block the generation of NICD. B) Western Blotting analysis of α–sma and MMP-2 expression in the cells treated with DAPT. C) Representative confocal immunofluorescence images cardiac fibroblasts treated with DAPT, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. D) Western blotting analysis of Jagged-1 expression in control cells, cells transfected with non specific scrambled-siRNA (SCR-siRNA) or silenced for the expression of Notch-1 ligand, Jagged-1, by specific Jagged-1 siRNA (Jagged-1 siRNA). E) Western Blotting analysis of α–sma in Jagged-1 silenced cells. F) Representative confocal immunofluorescence images cardiac fibroblasts silenced for Jagged-1 expression, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. The densitometric analyses of the bands normalized to GAPDH are reported in histograms in A–E; the densitometric analyses α–sma fluorescent signal are shown in the histograms in and C, F. Significance of differences: *p<0.05 vs control, δ p<0.05 vs SCR-siRNA, °p<0.05 vs TGF-β1, # p<0.05 vs TGF-β1+ RLX.

Article Snippet: To inhibit the expression of Jagged-1, short interference RNA duplexes (siRNA) (Santa Cruz) were used essentially as describred previously .

Techniques: Cell Culture, Western Blot, Expressing, Blocking Assay, Immunofluorescence, Staining, Transfection

Journal: PLoS ONE

Article Title: Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

doi: 10.1371/journal.pone.0063896

Figure Lengend Snippet: A) RT-PCR of Notch-1 expression. B,C) Western blotting analysis of activated intracellular form of Notch-1 (Notch-ICD, B) ) and of Notch-1 ligand, Jagged-1(C). The densitometric analyses of the bands normalized to GAPDH are reported in the histograms. D) Confocal immunofluorescence analysis of Notch-1 (green) and Hes-1 (cyan) expression. For the analysis of Notch-1, the cells were stained with a specific antibody recognizing both the membrane Notch-1 receptor and its activated intracellular form, Notch-ICD. Densitometric analyses of Notch-ICD and Hes-1 fluorescent signals are reported in the corresponding histogram. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β1.

Article Snippet: Primary neonatal cardiac fibroblasts (P1) were transfected using siRNA trasfection medium (Santa Cruz) with the mixed combination of Jagged-1 siRNA duplexes or with SCR-siRNA (20 nM) in serum and antibiotic free medium.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

doi: 10.1371/journal.pone.0063896

Figure Lengend Snippet: Neonatal cardiac fibroblasts were cultured for 48 h and treated as indicated. A) Western blotting analysis of NICD expression in the absence (control) or presence of DAPT (5 µM) a pharmacological γ-secretase inhibitor, used to block the generation of NICD. B) Western Blotting analysis of α–sma and MMP-2 expression in the cells treated with DAPT. C) Representative confocal immunofluorescence images cardiac fibroblasts treated with DAPT, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. D) Western blotting analysis of Jagged-1 expression in control cells, cells transfected with non specific scrambled-siRNA (SCR-siRNA) or silenced for the expression of Notch-1 ligand, Jagged-1, by specific Jagged-1 siRNA (Jagged-1 siRNA). E) Western Blotting analysis of α–sma in Jagged-1 silenced cells. F) Representative confocal immunofluorescence images cardiac fibroblasts silenced for Jagged-1 expression, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. The densitometric analyses of the bands normalized to GAPDH are reported in histograms in A–E; the densitometric analyses α–sma fluorescent signal are shown in the histograms in and C, F. Significance of differences: *p<0.05 vs control, δ p<0.05 vs SCR-siRNA, °p<0.05 vs TGF-β1, # p<0.05 vs TGF-β1+ RLX.

Article Snippet: Primary neonatal cardiac fibroblasts (P1) were transfected using siRNA trasfection medium (Santa Cruz) with the mixed combination of Jagged-1 siRNA duplexes or with SCR-siRNA (20 nM) in serum and antibiotic free medium.

Techniques: Cell Culture, Western Blot, Expressing, Blocking Assay, Immunofluorescence, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Protein analysis of Jagged1, TAZ, TEAD1, and Hes1 in A549 cell lysates after transfected with TAZ and concomitant silencing of TEAD1 by shRNA. b Hes1 reporter assay in the presence (+) or absence (−) of Notch1 ICD, or TAZ, N = 3. Results of luciferase reporter assays are shown. * P < 0.05. c TEAD1-reporter luciferase assay in the presence (+) or absence (−) of TAZ or nTEAD1, N = 3. d Jagged1 luciferase reporter assay in the presence (+) or absence (−) of Notch1 ICD, TAZ, or Mst1, N = 3. * P < 0.05. Cells were transfected with or without MST1, phosphorylated TAZ and TAZ were analyzed by western blot assay. e ChIP assay for Notch1 or TAZ in A549 cells transfected with Notch1, TAZ, and plasmid of either Jagged1-ECR1 or ECR6. Data are presented as fold enrichment over an IgG ChIP performed with the same samples. f ChIP assay for TAZ in A549 cells at Jagged1-ECR1, ECR6, and Hes1 promoter. N = 3. * P < 0.05.

Article Snippet: Jagged1 and TEAD1 were purchased from Santa Cruz Biotechnology (CA, US).

Techniques: Transfection, shRNA, Reporter Assay, Luciferase, Western Blot, Plasmid Preparation