Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Biological relevance of mutations in classical cadherin genes in skin cutaneous melanoma. ( A ) Lollipop plots of mutations in classical cadherin genes in skin cutaneous melanoma were presented by using NGS data from TCGA cohort (n = 448) ( left ) and the non-TCGA cohort (n = 396) ( right ). The non-TCGA cohort includes patients from Broad/Dana Farber (n = 26, Nature 2012), MSKCC (n = 64, NEJM 2014), Broad (n = 121, Cell 2012), Yale (n = 147, Nat Genet 2012), and UCLA (n = 38, Cell 2016). ( B ) Mutations in the intracellular domains of top-five cadherin genes were compared with mutations in CTNNB1 . ( C ) Co-occurrence analysis among top-four type-II classical cadherin genes (core CDHs) including CDH6, CDH9, CDH10, and CDH18. ( D ) Gene expression levels of core CDHs during melanoma malignant transition from normal neural crest cells and melanocytes to melanoma. Gene expression data were extracted from the GEO dataset (GDS1965). ( E ) Physical interactions (heterophilic/homophilic) among the top-four core CDHs were previously characterized and confirmed .

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Gene Expression

Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Impact of S524L mutation on the fifth extracellular (EC5) domain of the CDH6 protein. ( A ) Protein stability of wild type (WT) and mutant (S524L) CDH6 were compared by residue-specific Root-Mean-Square Deviation (RMSD) and Root-Mean-Square Fluctuations (RMSF) methods in simulations. The S524L mutation lead to higher instability in the β2/β3 loop and β4/β5 loop regions. ( B ) The EC5 structures of the WT and S524L CDH6 are drawn in cartoon putty representation, where the color is ramped by residue from blue as the lowest B-factor value to red as the highest B-factor value. In addition, the size of the tube also reflects the value of the B-factor, where the larger the B-factor the thicker the tube. The calcium atoms and mutation sites are indicated with gray sphere and yellow sphere representations, respectively. ( C ) The thermodynamic cycle for Ca 2+ binding affinity was calculated in one simulation box. The free energy difference in Ca 2+ binding corresponds to a double free energy difference: ΔΔG = ΔG1 - ΔG2.

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Thermo-stability of CDH6/catenin complex with mutations in the intracellular domain (CDH-C) of CDH6. ( A ) Alignment of CDH-C protein sequences among classical cadherins. Letters with underlines and yellow shadows indicate known mutations in the CDH-C sequences in skin cutaneous melanoma based on the NGS data from TCGA cohort. Letters with orange color represent the conserved residues whereas letters in red color represent identical residues among classical cadherins. Green box and red box indicate the regions of JMD for p120-catenin (δ-catenin) and CBD for β-catenin binding, respectively. ( B ) The complex structures of CDH6-JMD (blue)/p120-catenin (green) ( left ) and CDH6-CBD (blue)/β-catenin (green) ( right ) were predicted by using the MODELLER program. The mutation sites are highlighted in orange spheres. (C) The binding free energy differences (ΔΔG) caused by mutations in the domains of JMD for p120-catenin binding ( upper ) and CBD for β-catenin binding ( lower ) were calculated by using the fast-growth thermodynamic integration approach.

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Binding Assay, Mutagenesis

Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Functional impacts of mutations in the intracellular domain (CDH-C) of CDH6 on β-catenin binding. ( A ) Wild type (WT) CDH6 and its mutants (tagged with GFP) were co-transfected with β-catenin (tagged with mCherry) into A375 cells. Cells expressing both green and red constructs were monitored and representative images were taken under a fluorescent microscope 24 hrs after transfection. ( B ) Distributions of β-catenin in cells with different CDH6 constructs were counted and categorized into nucleus, cytoplasm and membrane (n = 50 cells for each CDH6 construct). ( C ) Dual-color immunofluorescence staining, CDH6 in red and β-catenin in green, was performed on melanoma tissue slides from patients with wild type cadherins and patients with D724N mutation in CDH6 . ( D ) Gene expression levels of genes involved in Hippo (blue box) and Wnt/β-catenin (pink box) pathways were compared between patients with mutations in top-four core cadherins (MT core CDHs) and patients without mutation in any classical cadherins (WT CDHs). The expression levels of indicated genes were extracted from the mRNA data of TCGA cohort. Statistical differences between two groups were compared by using t-test. The p values were presented as *: p value < 0.05, **: p value < 0.01, and ***: p value < 0.001.

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Functional Assay, Binding Assay, Transfection, Expressing, Construct, Microscopy, Membrane, Immunofluorescence, Staining, Mutagenesis, Gene Expression

Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Clinical significance of cadherin mutations during melanoma development. ( A ) Clinical associations were compared between patients with mutations in top-four core cadherins (MT core CDHs) and patients without any mutation in classical cadherin genes (WT CDHs), including tumor stage (left), lymph node invasion ( middle ), and metastasis ( right ). ( B ) Kaplan-Meier analyses were performed to compare overall survival ( upper ) and disease-free survival ( lower ) between MT core CDHs and WT CDHs groups. ( C ) Gene set enrichment analysis (GESA) was performed using mRNA expression data from TCGA group. Two pathways, T-lymphocyte-up ( upper ) and antigen response ( lower ) were found positively associated with mutations in top-four core CDH genes. ( D ) Lymphocyte scores in melanoma tissues of TCGA cohort were compared between MT core CDHs and WT CDHs groups. ( E ) Dual-color immunofluorescence staining, CD3 in red and β-catenin in green, was performed on melanoma tissue slides from patients with wild type cadherins and patients with K467R mutation in CDH6 ( left ). CD3 + T-lymphocytes in the stained tissue sections were quantified and compared between MT core CDHs and WT CDHs groups ( right ). For data in (A), (D) and (E), Fisher's exact test was performed to analyze the clinical association. For (B), the long-rank test was used to compare the survival times. The p values were presented as *: p value < 0.05, **: p value < 0.01, and ***: p value < 0.001.

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Mutagenesis, Expressing, Immunofluorescence, Staining

Journal: Theranostics

Article Title: Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development

doi: 10.7150/thno.46705

Figure Lengend Snippet: Immunogenicity of cadherin mutations and therapeutic advantages for treating melanoma. ( A ) The mutation counts (gray bars) for known CDH6 mutations in TCGA cohort were shown alone with the amino acid positions in CDH6. Antigen tolerance scores of the indicated mutation (red square) and its corresponding wild type (green circle) were analyzed by using NetMHCpan algorithm and plotted on this bar chart. Immunogenetic datasets of MSKCC and UCLA were utilized to compare ( B ) overall mutational loads; ( C ) Neo-antigen loads; ( D ) therapeutic duration times for Ipilimumab (anti-CTLA-4) treatment; and ( E ) drug responses for Pembrolizumab (anti-PD-1) therapy between MT core CDHs and WT CDHs groups. For data in (B) and (C), Fisher's exact test was performed to analyze the statistical significances. For (D) and (E), two-proportions Z-Test was utilized to compare drug effectiveness. The p values were presented as *: p value < 0.05, **: p value < 0.01, and ***: p value < 0.001.

Article Snippet: Human CDH6 cDNA (OriGene Technologies, Inc., USA) was amplified and sub-cloned into pAcGFP1-N1 vector (Clontech Lab.) to generate GFP-tagged wild type CDH6.

Techniques: Immunopeptidomics, Mutagenesis

Journal: PLoS ONE

Article Title: Cadherin-9 Is a Novel Cell Surface Marker for the Heterogeneous Pool of Renal Fibroblasts

doi: 10.1371/journal.pone.0000657

Figure Lengend Snippet: Sources of the used antibodies

Article Snippet: anti cadherin-6 (clone 2B6) , Acris antibodies (Hiddenhausen, Germany).

Techniques: Transduction

Journal: Nature Communications

Article Title: β-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis

doi: 10.1038/s41467-020-15332-7

Figure Lengend Snippet: a – c Colocalization of F-actin (green) and β-catenin (β-cat, magenta), N-cadherin (N-cad, magenta), and K-cadherin (K-cad, magenta) at rosette centers (white arrowheads) and smaller F-actin punctae (yellow arrowheads). DAPI (blue) marks nuclei. All bars, 10 μm. d Transmission electron micrographs showing adherens junctions in the zG. Left, red dashed line marks the boundary of a glomerulus. Red dots denote nuclei of zG cells. Red triangles denote nuclei of zF cells. Boxed area is shown at a higher magnification in the middle panel where individual cells are pseudo-colored. Red arrows point to adherens junctions. Right, an example of aggregating adherens junctions (red arrowheads). Bar sizes are indicated in each image.

Article Snippet: The following Taqman gene expression assays (Applied Biosystems) were used: Axin2 (Mm00443610_m1), Cdh2 (Mm01162497_m1), Cdh6 (Mm01310024_m1), Lef1 (Mm00550265_m1), Shroom3 (Mm00497207_m1), Ppib (Mm00478295_m1), Sulf1 (Mm00552283_m1), Nkd1 (Mm00471902_m1), Nfatc4 (Mm00452375_m1), Shh (Mm00436528_m1), Sema5a (Mm00436500_m1), Daam2 (Mm01273811_m1), Ajuba (Mm00495049_m1), Dact1 (Mm00458117_m1), Wnt4 (Mm01194003_m1), Dab2 (Mm01307290_m1), Gli3 (Mm00492337_m1), Tcf7 (Mm00493445_m1), Fzd5 (Mm00445623_s1), Lama5 (Mm01222029_m1), Prickle1 (Mm01297035_m1).

Techniques: Transmission Assay