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: Cell reports

Article Title: CDK1 bridges NF-κB and β-catenin signaling in response to H. pylori infection in gastric tumorigenesis

doi: 10.1016/j.celrep.2023.112005

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) in a mouse model with H. pylori infection was performed by comparing infection cases with non-infection cases. Canonical Wnt signaling pathway was significantly enriched in H. pylori infection samples (p < 0.01). (B) Pearson’s correlation test revealed strong correlations between CDK1 expression and CTNNB1 level in the GEO: GSE84433 cohort (R = 0.39, p < 0.01). (C) The immunoblot analyses were performed to determine β-catenin and p-GSK-3β (S9) expression in GES1 and AGS cells with overexpression of CDK1 or its knockdown by CDK1-specific siRNA in MKN45 cells. (D) Representative immunofluorescent images of β-catenin (green) and CDK1 (red) in AGS cells’ overexpression of CDK1; nuclei were stained with DAPI (blue). (E and F) β-catenin luciferase reporter assays. TOP-flash contains wild-type TCF binding sites. FOP-flash, containing mutated TCF binding sites, is served as a negative control; *p < 0.05 and **p < 0.01. (E) The gastric cancer (GC) cells were transfected with indicated amounts (0.5 or 1.0 μg) of CDK1 expression vector or empty vector control (Ctrl). (F) The MKN45 cells were transfected with CDK1 siRNAs or scrambled siRNA (Ctrl). (G and H) AGS or MKN28 cells were transfected with CDK1 expression plasmid or empty vector (Ctrl), and MKN45 cells were transfected with CDK1 siRNA or scramble siRNA control (Ctrl). Quantitative real-time PCR analysis of β-catenin targets, including AXIN2 and CCND1. Experiments were performed in triplicates. *p < 0.05 and **p < 0.01.

Article Snippet: TOP flash (WT TCF Reporter Plasmid) , upstate biotechnology , #21-170.

Techniques: Infection, Expressing, Western Blot, Over Expression, Knockdown, Staining, Luciferase, Binding Assay, Negative Control, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: CDK1 bridges NF-κB and β-catenin signaling in response to H. pylori infection in gastric tumorigenesis

doi: 10.1016/j.celrep.2023.112005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TOP flash (WT TCF Reporter Plasmid) , upstate biotechnology , #21-170.

Techniques: Virus, Recombinant, In Situ, Sample Prep, Plasmid Preparation, Mutagenesis, Software

Journal: Molecular and Cellular Biology

Article Title: Functional Differences of Very-Low-Density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling

doi: 10.1128/MCB.00235-16

Figure Lengend Snippet: A higher ectodomain shedding rate and a more potent Wnt pathway inhibitory effect of VLDLRII than VLDLRI in vitro . (A) CM and CLs were collected from CHO cells expressing GFP, VLDLRI, or VLDLRII. sVLDLR-N in CM and full-length VLDLR (FL-VLDLR) in CLs were detected using the 3D10 antibody. (B) The levels of the proteins were semiquantified by densitometry, and the ratios of sVLDLR-N to FL-VLDLR (normalized by the β-actin levels) were calculated and compared between VLDLRI and VLDLRII. (C) The total proteins in CM from cells expressing GFP (lane 1), VLDLRI (lane 2), or VLDLRII (lane 3) were analyzed by SDS-PAGE and stained by Coomassie blue. The band indicated by an arrow had a molecular weight identical to that of sVLDLR-N and existed only in CM from cells expressing VLDLRII. (D) CHO cells were separately infected with Ad-GFP, Ad-VLDLRI, or Ad-VLDLRII at the indicated MOIs. Similarly, sVLDLR-N and FL-VLDLR levels were measured by Western blotting. (E) Müller Top-Flash cells were treated with CM from CHO cells expressing VLDLRI or VLDLRII and Wnt3A CM for 16 h. The cells were then lysed, and TCF/β-catenin transcriptional activity was measured and normalized to that of control CM from cells expressing GFP. All data are representative of those from 3 independent experiments. Values are means ± SDs. P values were determined by Student's t test for two-group comparisons. **, P < 0.01; ***, P < 0.001; N.S., nonsignificant.

Article Snippet: Rat Müller Top-Flash cells were treated with specific CM and Wnt3A CM for 24 h, and a luciferase-based Wnt signaling activity assay (Top-Flash assay) was then conducted following the manufacturer's protocol (Promega, Madison, WI).

Techniques: In Vitro, Expressing, SDS Page, Staining, Molecular Weight, Infection, Western Blot, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Functional Differences of Very-Low-Density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling

doi: 10.1128/MCB.00235-16

Figure Lengend Snippet: VLDLRII has a more potent inhibitory effect on Wnt signaling than VLDLRI in vivo . (A and B) The levels of phosphorylated LRP6 (p-LRP6) and total LPR6 (t-LRP6) (A) and the levels of nonphosphorylated β-catenin (np-β-catenin) and total β-catenin (t-β-catenin) (B) in the eyecups of VLDLR −/− mice intravitreally injected with Ad-GFP, Ad-VLDLRI, or VLDLRII were measured by Western blotting. (C and D) Densitometry was performed to semiquantify p-LPR6 and t-LRP6 (C) and np-β-catenin and t-β-catenin (D), the levels of which were normalized by the β-actin levels. (E) Wnt signaling activity in the retina was evaluated by X-Gal staining of retinal sections from Axin2 lacZ /VLDLR +/+ (negative control), Axin2 lacZ /VLDLR −/− (positive control), and Axin2 lacZ /VLDLR −/− mice injected with adenovirus expressing VLDLRI (Ad-VLDLRI) and Axin2 lacZ /VLDLR −/− mice injected with adenovirus expressing VLDLRII (Ad-VLDLRII). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Magnifications, ×100. (F) The color intensities of the retinal section images were quantified and compared among the four groups shown in panel E. (G) Vascular endothelial cells in the retinal sections of VLDLR −/− mice injected with Ad-VLDLRI or Ad-VLDLRII were stained with lectin (green). The nuclei were counterstained with DAPI (blue). Magnifications, ×200. (H) The vascular areas (lectin staining shown in panel G) in the retinal sections were calculated and compared between VLDLR −/− mice injected with Ad-VLDLRI or Ad-VLDLRII. All data are representative of those from 3 independent experiments with 5 to 8 mice per group. Values are means ± SEMs. P values were determined by Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., nonsignificant.

Article Snippet: Rat Müller Top-Flash cells were treated with specific CM and Wnt3A CM for 24 h, and a luciferase-based Wnt signaling activity assay (Top-Flash assay) was then conducted following the manufacturer's protocol (Promega, Madison, WI).

Techniques: In Vivo, Injection, Western Blot, Activity Assay, Staining, Negative Control, Positive Control, Expressing

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