Fig 1: CDH11 disrupts the catabolism of BCAA leading to the activation of the mechanistic target of rapamycin pathway. A) The co‐immunoprecipitation assay demonstrates the interaction between CDH11 and BCKDHA, BCKDHB, and DBT proteins. B,C) Western blot showing increased phosphorylation levels of BCKDHE1α in CDH11‐overexpressed SACC‐83 cells. The results are reversed in CDH11‐knockdown cells. D) BCAA content in the culture medium shows no significant change after culturing SACC‐83 cells with CDH11 overexpression or knockdown for 24 h. n = 3, Mean ± SEM is shown. *P < 0.05 using t test. E) BCAA content per million SACC‐83 cells exhibits an increase following 24 h of CDH11‐overexpressed cell culture. The results are reversed in CDH11‐knockdown cells. n = 3, Mean ± SEM is shown, *P < 0.05 using t test. F) BCAA consumption per million SACC‐83 cells exhibits a decrease after culturing 24 h in CDH11‐overexpressed cells. The results are reversed in CDH11‐knockdown cells. n = 3, Mean ± SEM is shown, *P < 0.05 using t test. G) RNA‐seq analysis shows an upregulation of ATP6V1B1 and rnf152 gene expression and a downregulation of TNF gene expression in SACC‐83 cells with CDH11 overexpression. H) Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis shows that overexpressing CDH11 in cells significantly increased the expression of EMT‐associated transcription factors. The results are reversed in CDH11‐knockdown cells. n = 3, Mean ± SEM is shown, *P < 0.05 using t test. I) qRT‐PCR analysis reveals a significant upregulation of EMT‐associated genes in cells overexpressing CDH11. The results are reversed in CDH11‐knockdown cells. n = 3, Mean ± SEM is shown, *P < 0.05 using t test. J) The Dissociation Constants (KD) of Celecoxib (CXB), Dimethylcelecoxib (DMC) and SD‐133 with the CDH11 protein detected by Surface plasmon resonance were 4.67e‐06, 1.09e‐05, and 2.74e‐05 M, respectively.
Fig 2: Hybrid EMT cells are characterized by the upregulation of the gene expressions of PDGFRA, ITGA2, and CDH11. A) TheUMAP of epithelial subpopulations from both SMG and SACC labeled by cell type. The cell populations enclosed by dotted lines are hybrid EMT state cells. B) The cell number and percentage frequency of epithelial subpopulations in the scRNA‐seq data among different groups. C) The heatmap displays the expression levels of classical transcription factors that regulate EMT across different epithelial subpopulations. ZEB1, TWIST1, and SNAI1 have specific expression levels within the hybrid EMT state cell population. D) Dot plot showing marker gene distributions across the different epithelial subpopulations. E) Violin plot showing the expression of marker genes during hybrid EMT state cells, with PDGFRA, ITGA2, and CDH11 highly expressed in hybrid EMT state cells.
Fig 3: Metabolism of BCAA increases the production of ROS, activating the cGAS‐STING pathway. A) Flow cytometry detected ROS production in SACC‐83 cells with CDH11 knockdown. B) Representative images of DNA comet assays of SACC‐83 cells subjected to various experimental conditions. Scale bar, 20 µm. C,D) Confocal microscopy showing the accumulation and quantification of cytosolic DNA in SACC‐83 cells under knockdown CDH11. dsDNA visualized using PicoGreen staining (green), while MitoTracker (red) and DAPI (blue) employed to label mitochondria and nuclei, respectively. Scale bar, 5 µm. More than 100 cells were analyzed per group. Mean ± SEM is shown. n = 10, ***P < 0.001 using t test. E) Expression of cGAS‐STING pathway‐related proteins in SACC‐83 cells, treated under various experimental conditions, assessed using western blotting. F) Flow cytometry of SACC‐83 cells overexpressing CDH11 to evaluate the mitochondrial activity. G) Representative images of DNA comet assays of SACC‐83 cells subjected to various experimental conditions. Scale bar, 20 µm. H) Expression of cGAS‐STING pathway‐related proteins in SACC‐83 cells, treated under various experimental conditions, assessed using western blotting. I) qRT‐PCR analysis shows that CDH11‐knockdown cells significantly increase the expression of IFNB1. No significant difference in the overexpression group. n = 3, Mean ± SEM is shown, *P < 0.05 using t test. J) Flow cytometry detected ROS production in BCAA‐ deprived or ‐added SACC‐83 cells. K,L) Confocal microscopy showing the accumulation and quantification of cytosolic DNA in BCAA‐deprived or ‐added SACC‐83 cells. dsDNA visualized using PicoGreen staining (green), while MitoTracker (red) and DAPI (blue) employed to label mitochondria and nuclei, respectively. Scale bar, 5 µm. More than 100 cells were analyzed per group. n = 10, Mean ± SEM is shown. ***P < 0.001 using one‐way analysis of variance. M) The expression of cGAS‐STING pathway‐related proteins in SACC‐83 cells, treated under various experimental conditions, was assessed using western blotting.
Fig 4: CDH11 acts as a biomarker for hybrid EMT state cells, playing a crucial role in promoting cell proliferation, migration, invasion, and anoikis resistance. A) The UMAP shows the expression distribution of CDH11 in epithelial cells, and CDH11 is highly expressed in hybrid EMT state cells. B) Efficiency of CDH11 overexpressed or knocked down by a plasmid or small interfering RNA (siRNA) in SACC‐83 cells measured on western blotting. C,D) CDH11‐overexpressed SACC‐83 cells analyzed for their migration and invasion ability using transwell assays. The number of migrated and invaded cells was counted (n = 3, *P < 0.05). Scale bar, 200 µm, Mean ± SEM is shown, *P < 0.05 using t test. E,F) CDH11‐overexpressed or ‐knockdown SACC‐83 cells cultured for 0, 24, 48, and 72 h, and cell proliferation determined using the CCK‐8 assay. Mean ± SEM is shown, *P < 0.05 using t test. G,H) CDH11‐knockdown SACC‐83 cells analyzed for their migration and invasion ability using transwell assays. The number of migrated and invaded cells was counted (n = 3, *P < 0.05). Scale bar, 200 µm, Mean ± SEM is shown, *P < 0.05 using t test. I) CDH11‐overexpressed or ‐knockdown SACC‐83 cells cultured in low‐adhesion culture dishes, harvested after 48 h, and labeled with Annexin V‐FITC and PI were subjected to flow cytometry. J) Volcano plot showing differentially expressed genes in SACC‐83 cells after CDH11 overexpression, as identified via RNA‐seq. K) Top 10 terms of GO enrichment pathways in SACC‐83 cells after CDH11 overexpression.
Fig 5: Combination of CXB/DMC/SD‐133 with CDH11 activates the cyclic GMP‐AMP synthase‐stimulator of the interferon gene (cGAS‐STING) pathway through ROS, suppressing cellular invasion and migration. A) Representative western blots of the cellular thermal shift assay showing increase in CDH11 thermostable performance in the presence of CXB, DMC, and SD‐133. B) SD‐133 with interactive residue side chains at the pocket are shown in stick rendering, with the inhibitors drawn in colorful. The polypeptide backbones are rendered as ribbons. The yellow broken lines indicate potential intermolecular hydrogen bonds, while the gray broken lines indicate pi‐cation interactions. C–E) Effect of CXB, DMC, and SD‐133 on the inhibition of SACC‐83 cells; normalized data and non‐linear regression curve fitting are shown. IC50 values are indicated. F) BCAA per million SACC‐83 cells exhibited a decrease 24 h after treatment with CXB, DMC, or SD‐133. Mean ± SEM is shown, *P < 0.05 using t test. G) SACC‐83 cells treated with CXB, DMC, or SD‐133 analyzed for their migration and invasion ability using transwell assays. Scale bar, 100 µm, n = 3, Mean ± SEM is shown, *P < 0.05 using t test. H) Top 10 terms of GO enrichment analysis of DEG by RNA‐seq in SACC‐83 cells treated with SD133. I) Expression of cGAS‐STING pathway‐related proteins in SACC‐83 cells, treated with CXB, DMC, and SD 133, assessed using western blotting. J) Flow cytometry reveals an increase in reactive oxygen species (ROS) production after treatment with CXB, DMC, or SD‐133. K,L) Confocal microscopy showing the accumulation and quantification of cytosolic deoxyribonucleic acid (DNA) in SACC‐83 cells following treatment with CXB, DMC, or SD‐133. Double‐stranded DNA (dsDNA) visualized using PicoGreen staining (green), while MitoTracker (red) and DAPI (blue) employed to label mitochondria and nuclei, respectively. Scale bar, 5 µm. More than 100 cells were analyzed per group. n = 10, Means ± SEM is shown. ***P < 0.001 using one‐way analysis of variance. M) Representative images of DNA comet assays of SACC‐83 cells subjected to treatment with CXB, DMC, or SD‐133.
Supplier Page from Sino Biological, Inc. for Human Cadherin-11 Protein (His Tag)