Journal: Brain and Behavior

Article Title: E2F transcription factor 1 elevates cyclin D1 expression by suppressing transcription of microRNA‐107 to augment progression of glioma

doi: 10.1002/brb3.2399

Figure Lengend Snippet: E2F1 mediates the Wnt/β‐catenin signaling pathway. (a) Nuclear translocation of β‐catenin in A172 and T98G cells examined by immunofluorescence staining; (b) TOP/FOP activity in A172 and T98G cells determined by the TOP/FOP flash assay; (c) Protein levels of Wnt10B and β‐catenin in A172 and T98G cells detected by western blot analysis. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by one‐way ANOVA (b) or two‐way ANOVA (c), * p < .05 versus miR‐107 mimic +oe‐NC

Article Snippet: Cells in each group were cultured in 48‐well plates at a density of 2 × 10 4 cells per well for 24 h. Each well was loaded with 0.2 μg TOP/FOP flash and 1 ng Renilla (pRLTK) luciferase‐encoding plasmid (Promega) according to the instructions of a Lipofectamine 3000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Activity Assay, Western Blot

Journal: Molecular Cancer

Article Title: CircMYO10 promotes osteosarcoma progression by regulating miR-370-3p/RUVBL1 axis to enhance the transcriptional activity of β-catenin/LEF1 complex via effects on chromatin remodeling

doi: 10.1186/s12943-019-1076-1

Figure Lengend Snippet: CircMYO10 inhibits miR-370-3p to upregulate RUVBL1 expression and promotes the interaction between RUVBL1 and β-catenin/LEF1 complex and thus activates Wnt/β-catenin signaling. a MG63 cells were stably transfected with plasmids indicated while TOP/FOPFLASH plasmids, together with Renilla luciferase plasmids, were transiently transfected to measure the luciferase activity. Renilla luciferase activity was used as an internal control. Data represents the mean ± SD ( n = 9). b - c MG63 cells were stably transfected with expression vectors as indicated. Pre-miR-370-3p reduced the expression of RUVBL1 and β-catenin in MG63 cells. Lysates were incubated with either anti-β-catenin or anti-LEF1. Immunoprecipitated complexes were subjected to western blot analysis. Anti-RUVBL1 antibodies derived from different species were used to avoid the detection of heavy chains. d Western blot analysis of RUVBL1, β-catenin, C-myc and CyclinD1 in MG63 cells and U2OS cells stably transfected expression vectors as indicated. e Transfection with miR-370-3p sponge promoted the migration and invasion ability of both MG63 and U2OS cells which was partially abrogated by either ShRUVBL1 or TIP49D302N. Data represents the mean ± SD ( n = 3). (f) TCF/LEF luciferase activity assays were conducted in MG63 cells transfected with expression vectors indicated. Data represents the mean ± SD ( n = 9). g - h ShcircMYO10 downregulated the expression of g β-catenin, g RUVBL1 and h LEF1, which was restored by co-transfection with either RUVBL1 or miR-370-3p sponge in MG63 cells. Proteins immunoprecipitated with β-catenin either LEF1 were subjected to western blot analysis. i ShcircMYO10 decreases the expression of RUVBL1, β-catenin, cyclinD1, C-myc, N-cadherin and vimentin with E-cadherin upregulated, and the change was partially attenuated by co-transfection with either miR-370-3p sponge or RUVBL1. j The enhanced migration and invasion ability of MG63 and U2OS cells were partially abrogated by co-transfection with any of Pre-miR-370-3p or shRUVBL1 or TIP49D302N. Data represents the mean ± SD ( n = 3). Three independent assays were performed in the above assays. a , e , f , j * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test)

Article Snippet: TOPFLASH plasmids (GeneChem) contain 6 TCF/LEF binding sites which are mutated in FOPFLASH plasmids.

Techniques: Expressing, Stable Transfection, Transfection, Luciferase, Activity Assay, Control, Incubation, Immunoprecipitation, Western Blot, Derivative Assay, Migration, Cotransfection

Journal: Oncology Letters

Article Title: Inhibition of DNA methyltransferase 1 by RNA interference reverses epithelial-mesenchymal transition in highly metastatic 95D lung cancer cells by inhibiting the Wnt signaling pathway

doi: 10.3892/ol.2018.8449

Figure Lengend Snippet: Downregulation of DNMT1 inhibits the Wnt signaling pathway. (A) Relative luciferase activity representing β-catenin/TCF-dependent transcription in untreated 95Dcells (control) and 95D cells transfected with NC or DNMT1 siRNA. Cells were transfected with TOPflash or FOPflash along with pRL-SV40. FOPflash served as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity. Experiments were repeated three times. (B) Western blot analysis of cytosolic and nuclear β-catenin expression in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. (C) Western blot analysis of the expression of the β-catenin target cyclin D1 in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. GAPDH served as a loading control. **P<0.01, compared with the NC siRNA group. The results are representative of ≥3 independent experiments. DNMT1, DNA methyltransferase 1; siRNA, small interfering RNA; NC, negative control.

Article Snippet: 95D cells transfected with NC or DNMT1 siRNA were co-transfected with TOPflash or FOPflash plasmids (Upstate Biotechnology, Inc., Lake Placid, NY, USA), along with β-galactosidase expression plasmid pRL-SV40 (Promega Corporation, Madison, WI, USA), using Lipofectamine ® 2000 reagent.

Techniques: Luciferase, Activity Assay, Control, Transfection, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Heliyon

Article Title: Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

doi: 10.1016/j.heliyon.2024.e28488

Figure Lengend Snippet: The suppressive effect of EFNA4 on dCK depends on the β-catenin signaling activation. A, protein level of β-catenin in in BxPC-3/GEM and PANC-1/GEM cells after hyperthermia examined by WB analysis (uncropped images of blots are provided as Supplementary material); B, transcriptional activity of β-catenin in cells after hyperthermia determined by TOP Flash/FOP Flash luciferase reporter assay; C, transcriptional activity of β-catenin in cells after EFNA4 overexpression and MSAB treatment determined by TOP Flash/FOP Flash luciferase reporter assay; D, protein levels of β-catenin and dCK in cells after EFNA4 overexpression and MSAB treatment determined by WB analysis (uncropped images of blots are provided as Supplementary material). Three biological replicates were performed. Differences were compared by the two-way ANOVA followed by Sidak's (A–B) or Tukey's (C–D) multiple comparison test. * p < 0.05.

Article Snippet: To assess the β-catenin-mediated transcriptional activity of TCF/LEF, the TOP Flash reporter vector (D2501, Beyotime) was utilized, with the FOP Flash reporter vector containing mutant TCF/LEF binding sequences (D2503, Beyotime) serving as the NC.

Techniques: Activation Assay, Activity Assay, Luciferase, Reporter Assay, Over Expression, Comparison