Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation.

<p>(A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser⁹ GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser⁹ GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.</p

JNK2 deficiency caused upregulation of β-catenin and its downstream target CDK4, as well as upregulation of GSK3β phosphorylation in JNK2-/- mouse intestinal epithelial cells, compared to those in JNK2+/+ mice.

<p>Each lane represents one mouse. β-actin served as loading control.</p

Active JNK2 downregulated β-catenin expression and inhibited its transcriptional activity in a dose-dependent manner.

<p>(A) Activated JNK2 reduced β-catenin protein level in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin and p-JNK. β-actin served as loading control. (B) Activated JNK2 inhibited β-catenin-mediated transcriptional activity of TCF in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin, TOPFLASH, Renilla, along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.</p

Active JNK2-mediated β-catenin degradation occurred through the proteasome system and GSK3β.

<p>(A) HEK293T cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Forty-four hours after transfection, 25 µM MG132 was added to the indicated samples (lane 2 and 4). Four hours later cells were harvested for immunoblotting analysis to detect the expression of HA-β-catenin and p-JNK. (B) Blocking GSK3β activity by LiCl reduced β-catenin expression inhibition by activated JNK2. pcDNA3-HA-β-catenin was transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Thirty-six hours after transfection, half of the cultures were treated overnight with 30 mM LiCl (lane 2 and 4) and then harvested for immunoblotting analysis to detect the expression of HA-β-catenin, phospho-Ser-9 GSK3β, and p-JNK. (C) Mutant β-catenin was resistant to activated JNK2 induced degradation. Wild-type β-catenin (HA- β-catenin) (lanes 1 and 2) or various β-catenin mutants (HA-S33F β-catenin, lanes 3 and 4; HA-S33Y β-catenin, lanes 5 and 6; HA-S37A β-catenin, lanes 7 and 8) were transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 2,4,6,8) or empty vector (lanes 1,3,5,7). 48 hours after transfection, cells were harvested for immunoblotting analysis to determine the protein levels of HA-β-catenin. β-actin served as loading control.</p

Simulation_fidelity.m

This program is used to calculate the theoretical curve in the the Figure 2(a)

Activated JNK2 interacts with β-catenin and GSK3β.

<p>(A) Active JNK2 binding to β-catenin and GSK3β was analyzed by immunoprecipitation. β-catenin (HA tagged) was co-transfected with empty vector or active JNK2 (Flag tagged) into HEK293T cells. Immunoprecipitation was performed with a Flag antibody. (B) Mammalian two-hybridization assays showed a strong binding of β-catenin and JNK2 protein. The experiments were triplicated independently. (C) Active JNK2 and β-catenin co-localized in the cell nucleus and cytoplasm. Active JNK2 (Flag tagged) and pEGFP-β-catenin were co-transfected into HEK293T cells. The cells were immunostained with a Flag antibody. Co-localization (yellow fluorescence) of active JNK2 (red fluorescence) and β-catenin (green fluorescence) was detected in the nucleus and cytoplasm.</p

Table_1_Integrated analysis of anti-tumor roles of BAP1 in osteosarcoma.docx

BackgroundThis study aims to screen out differentially expressed genes (DEGs) regulated by BRCA1-associated protein 1 (BAP1) in osteosarcoma cells, and to analyze their biological functions.MethodsThe microarray dataset GSE23035 of BAP1-knockdown osteosarcoma cells was obtained from Gene Expression Omnibus (GEO) database, consisting of shControl, shBAP1#1 and shBAP1#2 samples. The DEGs between the BAP1-knockdown osteosarcoma cells and the untreated osteosarcoma cells were screened with limma package, and then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Gene Set Enrichment Analysis (GSEA) was also performed for the three groups of samples. Hub genes in a protein-protein interaction (PPI) network of DEGs was filtered, and then subjected to prognostic analysis and correlation analysis with BAP1 in Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Besides, the correlation between BAP1 and biological processes/pathways was analyzed by Gene Set Variation Analysis (GSVA) method and the correlation between BAP1 and immune infiltration by CIBERSORT and ESTIMATE methods. The roles of BAP1 in regulating proliferation and epithelial-mesenchymal transition (EMT) were validated by CCK-8 and western blot.Results58 upregulated DEGs and 81 downregulated DEGs were obtained with |logFC| ≥ 1 and adj.p ConclusionBAP1 might be a tumor suppressor in osteosarcoma and a promising therapeutic target.</p

Image_3_Integrated analysis of anti-tumor roles of BAP1 in osteosarcoma.tif

BackgroundThis study aims to screen out differentially expressed genes (DEGs) regulated by BRCA1-associated protein 1 (BAP1) in osteosarcoma cells, and to analyze their biological functions.MethodsThe microarray dataset GSE23035 of BAP1-knockdown osteosarcoma cells was obtained from Gene Expression Omnibus (GEO) database, consisting of shControl, shBAP1#1 and shBAP1#2 samples. The DEGs between the BAP1-knockdown osteosarcoma cells and the untreated osteosarcoma cells were screened with limma package, and then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Gene Set Enrichment Analysis (GSEA) was also performed for the three groups of samples. Hub genes in a protein-protein interaction (PPI) network of DEGs was filtered, and then subjected to prognostic analysis and correlation analysis with BAP1 in Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Besides, the correlation between BAP1 and biological processes/pathways was analyzed by Gene Set Variation Analysis (GSVA) method and the correlation between BAP1 and immune infiltration by CIBERSORT and ESTIMATE methods. The roles of BAP1 in regulating proliferation and epithelial-mesenchymal transition (EMT) were validated by CCK-8 and western blot.Results58 upregulated DEGs and 81 downregulated DEGs were obtained with |logFC| ≥ 1 and adj.p ConclusionBAP1 might be a tumor suppressor in osteosarcoma and a promising therapeutic target.</p

Image_1_Integrated analysis of anti-tumor roles of BAP1 in osteosarcoma.tif

BackgroundThis study aims to screen out differentially expressed genes (DEGs) regulated by BRCA1-associated protein 1 (BAP1) in osteosarcoma cells, and to analyze their biological functions.MethodsThe microarray dataset GSE23035 of BAP1-knockdown osteosarcoma cells was obtained from Gene Expression Omnibus (GEO) database, consisting of shControl, shBAP1#1 and shBAP1#2 samples. The DEGs between the BAP1-knockdown osteosarcoma cells and the untreated osteosarcoma cells were screened with limma package, and then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Gene Set Enrichment Analysis (GSEA) was also performed for the three groups of samples. Hub genes in a protein-protein interaction (PPI) network of DEGs was filtered, and then subjected to prognostic analysis and correlation analysis with BAP1 in Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Besides, the correlation between BAP1 and biological processes/pathways was analyzed by Gene Set Variation Analysis (GSVA) method and the correlation between BAP1 and immune infiltration by CIBERSORT and ESTIMATE methods. The roles of BAP1 in regulating proliferation and epithelial-mesenchymal transition (EMT) were validated by CCK-8 and western blot.Results58 upregulated DEGs and 81 downregulated DEGs were obtained with |logFC| ≥ 1 and adj.p ConclusionBAP1 might be a tumor suppressor in osteosarcoma and a promising therapeutic target.</p

Measuring and Modeling the Density of Some Typical Binary Mixtures in the Conditions of 280–423.15 K and 0.1–9 MPa

In order to investigate the interactive effect on the density of two-component mixtures, six mixtures of methylcyclohexane(1) + n-dodecane(2)/n-tetradecane(2)/n-hexadecane(2) and ethylcyclohexane(1) + n-dodecane(2)/n-tetradecane(2)/n-hexadecane(2) were measured by using the Anton Paar DMA 4200 M densimeter and a hand-operated pressure pump in the temperature and pressure ranges of T = 280 to 423.15 K and P = 0.1 to 9 MPa, respectively. Additionally, the combined standard uncertainty in the density of binary mixtures with n-hexadecane is uc(ρ) = 1.543 kg/m3, and for other mixtures, it is uc(ρ) = 1.541 kg/m3. The experimental data were successfully correlated with the empirically modified Toscani–Szwarc equation of state with a maximum regression deviation of less than 1.0630 kg/m3 for all the binary mixtures. On the basis of the experimental data and parameters of the regression model, isobaric thermal expansivity and isothermal compressibility were calculated and exhibited nonlinear behavior against the mole fraction, which is due to different molecular interactions between the two components