Journal: Science advances

Article Title: A claudin5-binding peptide enhances the permeability of the blood-brain barrier in vitro.

doi: 10.1126/sciadv.adq2616

Figure Lengend Snippet: Fig. 5. Assessment of the mCLDN5-peptide binding affinity. (A) Representative confocal imaging of HEK-293T cells transfected with a tGFP-tagged mCLDN5 construct and subjected to immunocytochemistry with mCLDN5 antibodies. Nuclei are in cyan, GFP in yellow, and mCLDN5 in pink. Scale bars, 20 μm. (B) MST binding curves and extracted Kd for f1-C5C2 (red symbols) and f3-cCPE–mut1 (green symbols) peptides, together with a negative control sample (black symbols, recombinant tGFP spotted in the cell lysate in the absence of transfected mCLDN5). The extracted Kd value for f1-C5C2 was 68.26 μM.

Article Snippet: The total GFP amount was estimated through a calibration curve obtained by measuring the fluorescence intensity of the recombinant tGFP protein (Pontellina plumata, catalog no.: TP700079, Origene) at increasing concentrations with a Tecan SPARK (Tecan, Switzerland) multimode microplate reader.

Techniques: Binding Assay, Imaging, Transfection, Construct, Immunocytochemistry, Negative Control, Recombinant

Journal: Frontiers in Oncology

Article Title: Tumor Suppressive Role of MUC6 in Wilms Tumor via Autophagy-Dependent β-Catenin Degradation

doi: 10.3389/fonc.2022.756117

Figure Lengend Snippet: MUC6 reduces β-catenin protein level. (A) Immunoblots showing protein levels of MUC6, β-catenin, c-Myc, Survivin and MMP7 in HEK293 cell infected with indicated shRNAs. (B) Real-time PCR results showing the mRNA levels of MUC6 and β-catenin in HEK293 cell infected with indicated shRNAs. (C) Immunoblots showing protein levels of MUC6, β-catenin, c-Myc, Survivin and MMP7 in WiT49 cell infected with indicated shRNAs. (D) Real-time PCR results showing the mRNA levels of MUC6 and β-catenin in WiT49 cell infected with indicated shRNAs. (E) Real-time PCR results showing β-catenin mRNA levels in 12 pairs of WT tissues. (F) Luciferase assays showing the activity of β-catenin promoter in cells infected with indicated shRNAs. (G) Luciferase assays showing the activity of β-catenin 3’UTR in cells infected with indicated shRNAs. (H) Immunoblots showing protein levels of β-catenin, c-Myc, Survivin and MMP7 after over-expression of MUC6-N or C terminal in HEK293 and WiT49 cell lines. (I) Real-time PCR results showing β-catenin mRNA levels after MUC6-C over-expression in HEK293 and WiT49 cell lines. (J) Luciferase assays showing the activity of β-catenin promoter in HEK293 and WiT49 cell lines after MUC6-C over-expression. (K) Luciferase assays showing the activity of β-catenin 3’UTR in HEK293 and WiT49 cell lines after MUC6-C over-expression. For all, *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: CRISPR-mediated editing in WiT49 cell was performed with pLenti-Cas9-P2A-tGFP from Origene and gRNAs targeting indicated sites were cloned into this vector.

Techniques: Western Blot, Infection, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Over Expression

Journal: Frontiers in Oncology

Article Title: Tumor Suppressive Role of MUC6 in Wilms Tumor via Autophagy-Dependent β-Catenin Degradation

doi: 10.3389/fonc.2022.756117

Figure Lengend Snippet: Autophagy pathway mediates MUC6-induced β-catenin degradation in WT cell. (A) Immunoblots showing β-catenin protein levels in WiT49 cell treated with Z-VAD-FMK, Ac-AEVD-CHO, MG-101, Calpeptin, MG132, Lactacystin, 3-MA or Bafilomycin A1 at indicated concentrations. (B) Immunoblots showing β-catenin protein levels in WiT49 cell infected with MUC-C alone or with dominant negative Atg5 together. (C) Immunoblots showing the effects of MUC-N or MUC-C over-expression on Atg5, Atg7, Beclin-1 and LC3A/B protein levels in WiT49 cell infected with MUC shRNA-1. For all, *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: CRISPR-mediated editing in WiT49 cell was performed with pLenti-Cas9-P2A-tGFP from Origene and gRNAs targeting indicated sites were cloned into this vector.

Techniques: Western Blot, Infection, Dominant Negative Mutation, Over Expression, shRNA

Journal: Frontiers in Oncology

Article Title: Tumor Suppressive Role of MUC6 in Wilms Tumor via Autophagy-Dependent β-Catenin Degradation

doi: 10.3389/fonc.2022.756117

Figure Lengend Snippet: MUC6 mutations compromise autophagy-dependent β-catenin degradation. (A) Immunoblots showing MUC6 protein levels in two clones from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. (B) Immunoblots showing β-catenin, c-Myc, Survivin and MMP7 protein levels in two clones from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. (C) Real-time PCR results showing β-catenin mRNA levels in two clones from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. (D) Luciferase assays showing the activity of β-catenin promoter in two clones from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. (E) Luciferase assays showing the activity of β-catenin 3’UTR in two clones from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. (F) Immunoblots showing the effects of MUC-N or MUC-C over-expression on Atg5, Atg7, Beclin-1 and LC3A/B protein levels in one clone from isogenic control, T1544K mutant and P1873Q mutant WiT49 cells, respectively. For all, **P<0.01.

Article Snippet: CRISPR-mediated editing in WiT49 cell was performed with pLenti-Cas9-P2A-tGFP from Origene and gRNAs targeting indicated sites were cloned into this vector.

Techniques: Western Blot, Clone Assay, Control, Mutagenesis, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Over Expression

Journal: Frontiers in Oncology

Article Title: Tumor Suppressive Role of MUC6 in Wilms Tumor via Autophagy-Dependent β-Catenin Degradation

doi: 10.3389/fonc.2022.756117

Figure Lengend Snippet: MUC6-autophagy-β-catenin regulates tumor behaviors in in-vitro assays. (A) MTT assay showing the proliferation of WiT49 cell infected with indicated vectors. (B) Transwell invasion assay showing the invasion of WiT49 cell infected with indicated vectors. (C) Annexin V apoptosis assay showing the apoptosis of WiT49 cell infected with indicated vectors. (D) MTT assay showing the proliferation of one clone from isogenic control cell and P1873Q mutant WiT49 cell, respectively. (E) Transwell invasion assay showing the invasion of one clone from isogenic control cell and P1873Q mutant WiT49 cell, respectively. (F) Annexin V apoptosis assay showing the apoptosis of one clone from isogenic control cell and P1873Q mutant WiT49 cell, respectively. For all, *P<0.05; **P<0.01; ***P<0.001. (G) Representative images of xenografted model and quantification of tumor size of three groups: isogenic control WiT49 cell (n=5), MUC6 P1873Q mutant WiT49 cell (n=5) and MUC6 P1873Q mutant WiT49 cell plus DN-β-catenin (n=5). **P<0.01.

Article Snippet: CRISPR-mediated editing in WiT49 cell was performed with pLenti-Cas9-P2A-tGFP from Origene and gRNAs targeting indicated sites were cloned into this vector.

Techniques: In Vitro, MTT Assay, Infection, Transwell Invasion Assay, Apoptosis Assay, Control, Mutagenesis