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: Experimental and Therapeutic Medicine

Article Title: miR-140-3p enhances cisplatin sensitivity and attenuates stem cell-like properties through repressing Wnt/β-catenin signaling in lung adenocarcinoma cells

doi: 10.3892/etm.2020.8847

Figure Lengend Snippet: miR-140-3p represses Wnt/β-catenin signaling in LUAD. mRNA expression levels of (A) total β-catenin, (B) c-Myc and (C) cyclin D1 in A549 and Calu3 cells following transfection with miR-140-3p or the control mimic. (D) Protein expression levels of total β-catenin, c-Myc and cyclin D1 following transfection with miR-140-3p or the control mimic. (E) TOP/FOP flash activity in A549 and Calu3 cells following transfection with miR-140-3p or the control mimic. Data are presented as the mean ± standard deviation from three independent experiments. * P<0.05 and ** P<0.01 vs. control mimic. miR, microRNA; LUAD, lung adenocarcinoma.

Article Snippet: Subsequently, cells were co-transfected with 100 ng TOP/FOP flash vector (Promega Corporation), internal control pRL-TK Renilla luciferase vector (10 ng) and control mimic or miR-140-3p mimics (50 nM) using Lipofectamine ® 2000.

Techniques: Expressing, Transfection, Activity Assay, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: miR-140-3p enhances cisplatin sensitivity and attenuates stem cell-like properties through repressing Wnt/β-catenin signaling in lung adenocarcinoma cells

doi: 10.3892/etm.2020.8847

Figure Lengend Snippet: miR-140-3p attenuates the stem cell-like properties in LUAD by suppressing Wnt/β-catenin signaling. (A) Protein and (B) mRNA expression levels of β-catenin in A549 and Calu3 cells following transfection with either the empty vector or vector expressing β-catenin. ** P<0.01 vs. vector. (C) Protein levels of total and nuclear β-catenin, * P<0.05 vs. vector + miR-140-3p mimic; ** P<0.01 vs. vector + control mimic. (D) TOP/FOP flash activity, * P<0.05 vs. vector + miR-140-3p mimic, ** P<0.01 vs. vector + control mimic. (E) number of spheres with diameter >100 µm formed by A549 and Calu3 cells. Scale bar, 100 µm. * P<0.05 vs. vector + miR-140-3p mimic, ** P<0.01 vs. vector + control mimic. (F) percentage of CD133 + cells analyzed using flow cytometry following co-transfection with either the empty vector or vector expressing β-catenin and miR-140-3p mimic or the control mimic. * P<0.05 vs. vector + miR-140-3p mimic, ** P<0.01 vs. vector + control mimic. Data are represented the mean ± standard deviation from three independent experiments. miR, microRNA; LUAD, lung adenocarcinoma; CD133, cluster of differentiation 133; ctnnb1 , β-catenin gene.

Article Snippet: Subsequently, cells were co-transfected with 100 ng TOP/FOP flash vector (Promega Corporation), internal control pRL-TK Renilla luciferase vector (10 ng) and control mimic or miR-140-3p mimics (50 nM) using Lipofectamine ® 2000.

Techniques: Expressing, Transfection, Plasmid Preparation, Activity Assay, Flow Cytometry, Cotransfection, Standard Deviation

Journal: Aging (Albany NY)

Article Title: METTL14 benefits the mesenchymal stem cells in patients with steroid-associated osteonecrosis of the femoral head by regulating the m6A level of PTPN6

doi: 10.18632/aging.203778

Figure Lengend Snippet: PTPN6-mediated METTL14 activated the Wnt signaling pathway. ( A ) The activity of Wnt signaling in SONFH BMSCs was analyzed by TOP/FOP-Flash reporter assay. ( B ) The protein level of β-catenin in the nucleus was measured by western blot. ( C ) Western blot detected the total and phosphorylated β-catenin levels in SONFH BMSCs treated as indicated. ( D ) Immunofluorescence staining was used to analyze the distribution of β-catenin in the cytoplasm and nucleus after METTL14 overexpression and PTPN6 knockdown. Scale bar = 50 μm. ( E ) The interaction of PTPN6 and GSK-3β in BMSCs was verified by co-IP assay. ( F ) The effect of PTPN6 inhibition on the phosphorylation level of GSK-3β in SONFH BMSCs was determined by western blot. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s is no significance.

Article Snippet: BMSCs were seeded in 24-well plates and transfected with the TOP/FOP-Flash reporter (Biovector NTCC Inc., China).

Techniques: Activity Assay, Reporter Assay, Western Blot, Immunofluorescence, Staining, Over Expression, Co-Immunoprecipitation Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: MMP7 promoted EMT in an E-cadherin/β-catenin pathway-dependent manner. ( A ) Protein expression of E-cadherin in HIBEpiCs after treatment with different concentrations of MMP7 for 24 h. ( B ) Level of sE-cadherin in the cell culture supernatant of HIBEpiCs after MMP7 treatment. ( C ) Western blot analysis of β-catenin protein in the nucleus or cytoplasm of HIBEpiCs. ( D ) Immunofluorescence staining shows the subcellular localization of β-catenin in HIBEpiCs. Scale bar:50 μm. ( E ) TOP/FOP-Flash luciferase activity of MMP7-treated cells. ( F ) Effect of the MMP7 and ICG-001 on β-catenin downstream target genes expression (including SNAI1 , SNAI2 , MMP7 ) in HIBEpiCs. ( G , H ) Effect of the MMP7 and ICG-001 on mRNA and protein expression of EMT-related markers (including E-cadherin, Vimentin, S100A4) in HIBEpiCs. ( I ) ELISA analysis of serum level of sE-cadherin in patients. ( J ) Spearman’s rank correlation analysis of serum sE-cadherin and serum MMP7 in BA patients. ( K ) Representative images of immunostaining of β-catenin in cholangiocytes of non-BA and BA patients. Magnification: left panel : ×100; right panel : ×400. ( C – H ) MMP7 concentration: 80 ng/mL; ICG-001 concentration: 25 μM. Data are presented as mean ± SD.

Article Snippet: The TOP/FOP Flash test was conducted following the guidelines provided by the manufacturer. (Upstate Biotechnology, Lake Placid, NY, USA).

Techniques: Expressing, Cell Culture, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Concentration Assay