Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) RON M1254T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in MDCK cells. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4, and 24 h later these cells were transfected with luciferase reporter plasmids containing WT (TOPFLASH) or mutated (FOPFLASH) Tcf promoter. Tcf activity was determined by a luciferase assay as described in Materials and Methods. Data were normalized for total protein concentration. Data for a negative control FOPFLASH are not shown. Luciferase activity was calculated in fold increase, where luciferase activity in cells expressing RON WT and β-Gal was taken for 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated RON is mediated by Tcf. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting (WB) with anti-c-myc and anti-cyclin D1 antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Expressing, Infection, Transfection, Luciferase, Activity Assay, Protein Concentration, Negative Control, Western Blot, Molecular Weight

Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) MET M1268T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in NIH 3T3 cells. Activity of Tcf-4 in NIH 3T3 cells expressing MET WT or M1268T was determined by luciferase assay as described in the legend to Fig. ​Fig.6A.6A. Luciferase activity in cells expressing MET WT and β-Gal was set equal to 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated MET is mediated by Tcf. NIH 3T3 cells expressing MET WT or M1268T were infected with an adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting with anti-c-myc and anti-cyclin D1 antibodies. MET tyrosine phosphorylation was determined by anti-PY antibodies in MET IPs. To estimate the amount of the MET in precipitates, the blot was probed with anti-MET antibodies (upper band, immature MET [170 kDa]; lower band, mature MET [140 kDa]). Tyrosine phosphorylation of β-catenin was detected by Western blotting (WB) with anti-PY antibodies. The amount of β-catenin in precipitates was determined with anti-β-catenin antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Activity Assay, Expressing, Luciferase, Infection, Western Blot, Molecular Weight

Journal: Cell reports

Article Title: Initiation of fibronectin fibrillogenesis is an enzyme-dependent process

doi: 10.1016/j.celrep.2023.112473

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Oxidation site 2 mutation (reverse): TAACACGTTGCCTCATGAG , Metabion (HyLabs) , N/A.

Techniques: Virus, Recombinant, Clinical Proteomics, Mutagenesis, Software, Microscopy, Imaging, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: DYNLT1 is a direct downstream miR-15b-3p target in GC cells. a miR-15b-3p target gene prediction using four bioinformatics tools (miRDB, RNA22, TarBase and TargetScan). b DYNLT1 mRNA levels in GC tissues and paired adjacent non-GC tissues ( n = 108) analyzed using qRT-PCR. c GC tissue DYNLT1 expression was found to be significantly decreased, based on the TCGA database. d-f Western blotting and IHC analysis of DYNLT1 protein levels in GC tissues and adjacent non-GC tissues. Scale bar, 200 μm. g miR-15b-3p and DYNLT1 expression level association analysis using the 108 GC tissues. h-i Immunoblotting assays and qRT-PCR on DYNLT1 expression of miR-15b-3p inhibitor/inhibitor-NC/mimics/NC transfected BGC-823 and SGC-7901 cells. j DYNLT1 binding site of the wild-type (WT) and mutated type with miR-15b-3p. k Direct recognition of DYNLT1 3′-UTR by miR-15b-3p. Co-transfection of BGC-823 and SGC-7901 cells with WT or Mutant DYNLT1 3′-UTR and miR-15b-3p mimics, inhibitor or their corresponding normal control (NC or inhibitor-NC). The relative luciferase activity of BGC-823 and SGC-7901 cells were determined. Mean ± SEM of the results are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Binding Assay, Cotransfection, Mutagenesis, Control, Luciferase, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Exosomal transfer of miR-15b-3p enhances malignant transformation in vitro. Proliferation of SGC-7901 and GES-1 cells co-cultured with PBS alone or exosomes containing miR-15b-3p mimics/NC/inhibitor/inhibitor-NC were assessed using colony formation ( a ), CCK-8 ( b ) and EdU ( c ) assays. Scale bar, 100 μm. d Migration and invasion assays of SGC-7901 and GES-1 cell treated with PBS, Exo-NC, Exo-15b-3p-mimics, Exo-inhibitor-NC or Exo-15b-3p-inhibitor. Cells that had migrated and invaded were counted. Representative images are shown. e The SGC-7901 and GES-1 cell apoptosis, in the presence of PBS or exosomes (wrapped with 15b-3p-mimics, inhibitor or their corresponding normal control) were detected using flow cytometry. f miR-15b-3p mimics/NC/inhibitor/inhibitor-NC-enriched exosomes or only PBS was incubated with GES-1 and SGC-7901 cells for 24 h, followed by TUNEL assay. g Western blotting assay of DYNLT1, BAX, BCL-2, Cleaved Caspase-9 and Cleaved Caspase-3 in SGC-7901 and GES-1 with the treatments indicated. The internal control used was β-Actin. Mean ± SEM of three independent experiments are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Transformation Assay, In Vitro, Cell Culture, CCK-8 Assay, Migration, Control, Flow Cytometry, Incubation, TUNEL Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Exo-miR-15b-3p regulates tumor growth in vivo. a miR-15b-3p expression levels in BGC-823 cells (stably transfected with Lv-miR-15b-3p/Lv-NC or Lv-inhibitor/Lv-inNC) or exosomes isolated from BGC-823 cells were detected using qRT-PCR. SGC-7901 cells were treated with PBS or exosomes loaded with Lv-miR-15b-3p/Lv-NC or Lv-inhibitor/Lv-inNC and were subsequently injected into the nude mice ( n = 5). The xenografts ( b) and tumor growth curve ( c) show that Exo-Lv-miR-15b-3p promotes, while Exo-Lv-inhibitor suppresses xenograft tumor growth in nude mice. d Representative images of tumor growth of the mice treated with exosomes derived from stably transfected-BGC-823 cells or PBS, were determined using luciferase-based bioluminescence imaging. e Representative images of TUNEL staining of the xenograft tumors for the ectopic expression or silencing of Exo-miR-15b-3p and their corresponding control or PBS groups. Scale bar, 100 μm. f Quantification of TUNEL-positive cells. g qRT-PCR analysis of miR-15b-3p and DYNLT1 expression in xenograft tumors with the treatment indicated. h Immunohistochemical analysis of DYNLT1 expression in the xenografts. Scale bar, 50 μm. i Western blotting analysis of DYNLT1, BAX, BCL-2, Cleaved Caspase-9 and Cleaved Caspase-3 in xenograft tumor tissues among the different groups. The internal control used was β-Actin. Mean ± SEM of the results are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: In Vivo, Expressing, Stable Transfection, Transfection, Isolation, Quantitative RT-PCR, Injection, Derivative Assay, Luciferase, Imaging, TUNEL Assay, Staining, Control, Immunohistochemical staining, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Schematic illustrating the function and mechanism of GC cells-derived exo-miR-15b-3p in recipient cells. The exo-miR-15b-3p released by GC cells promotes the proliferation, migration, invasion and inhibits apoptosis of recipient cells via the DYNLT1/Caspase-3/Caspase-9 signaling pathway, thereby promoting tumorigenesis and malignant transformation. Serum exo-miR-15b-3p may function as a potential GC diagnostic biomarker

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Derivative Assay, Migration, Transformation Assay, Diagnostic Assay, Biomarker Discovery