Journal: Cells

Article Title: Metastatic Tumor Cell-Specific FABP7 Promotes NSCLC Metastasis via Inhibiting β-Catenin Degradation

doi: 10.3390/cells11050805

Figure Lengend Snippet: FABP7 is highly expressed in NSCLC cells. ( A ) UMAP visualization of all cells in the single-cell dataset colored by identified cell types. ( B ) UMAP visualization of NK and T cells (top) and myeloid cells (bottom) in the single-cell dataset colored by identified cell types. ( C ) Identification of tumor cells. Inferred copy number variations (CNVs) patterns of each chromosome (shown by different color bars) were compared between normal cells (Reference Cells) and epithelial cells from tumor tissues (Observation Cells). A higher inferCNV score (right) represents a higher CNVs degree of the cluster. The red dash line box shows malignant cells with obvious CNVs and higher inferCNV scores. ( D ) UMAP visualization of tumor cells from non-metastatic (top) or metastatic (bottom) patients. ( E ) Volcano plot of differentially expressed genes (DEGs) between tumor cells of metastatic and non-metastatic patients. Expressions with adjusted p -value < 0.05 are shown in red (log 2 Fold change > 1) or blue (log 2 Fold change < −1), while those with adjusted p -value > = 0.05 are shown in grey. ( F ) FABP7 Expression of all main cell types (left) or tumor cells (right) split by patients with or without metastasis. Expression levels of FABP7 are labeled by the intensity of red color. ( G ) Average expressions of FABP3-7 in indicated cell types split by patients without (left) or with (right) metastasis. ( H ) Kaplan-Meier overall survival (OS) analyses of FABP3-7 in NSCLC based on KM-plotter.

Article Snippet: Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively.

Techniques: Expressing, Labeling

Journal: Cells

Article Title: Metastatic Tumor Cell-Specific FABP7 Promotes NSCLC Metastasis via Inhibiting β-Catenin Degradation

doi: 10.3390/cells11050805

Figure Lengend Snippet: FABP7 contributes to NSCLC metastasis and indicates poor prognoses. ( A ) Metastasis-related biological functions enriched by Gene Ontology (GO) analysis. DEGs from scRNA-seq comparing FABP7-positive with FABP7-negative tumor cells were used for the analysis. ( B ) Gene Set Enrichment Analysis (GSEA) for DEGs between FABP7-positive and FABP7-negative malignant cells from scRNA-seq dataset. ( C ) Expression efficiencies of FABP7 stable over-expression (left) and knockdown (right) cell lines. Representative images from three independent experiments are shown. ( D ) Western blot analyses of EMT markers in NSCLC cells with stable over-expression or knockdown of FABP7. Representative images from three independent experiments are shown. ( E ) NSCLC cells were plated in chambers without or with matrigel respectively to perform transwell assays. Migratory (without matrigel) or invasive cells (with matrigel) were quantified in five random fields. Scale bar: 50 μm. Error bars represent the means ± SD derived from three independent experiments. Statistical analyses were performed by two-sided Mann-Whitney test (*: p < 0.05). ( F ) Representative H&E staining images (insets: high-magnification images) of xenografts caused by vector or FABP7 A549 cells. n = 4, Scale bar: 50 μm. ( G , H ) Indicated A549 cells (1 × 10 6 ) were injected via the tail vein. Representative bioluminescent images ( G ), picric acid staining and H&E staining ( H ) of lung metastases are shown. n = 5, Scale bar: 50 μm. ( I ) Metastasis-free survival (MFS) Kaplan-Meier analyses of LUAD from the MSKCC dataset.

Article Snippet: Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively.

Techniques: Expressing, Over Expression, Western Blot, Derivative Assay, MANN-WHITNEY, Staining, Plasmid Preparation, Injection

Journal: Cells

Article Title: Metastatic Tumor Cell-Specific FABP7 Promotes NSCLC Metastasis via Inhibiting β-Catenin Degradation

doi: 10.3390/cells11050805

Figure Lengend Snippet: FABP7 transactivates Wnt/β-catenin signaling. ( A ) Wnt signaling pathway enriched by GO analysis. DEGs from scRNA-seq comparing between FABP7-positive and FABP7-negative tumor cells were analyzed. ( B ) GSEA for Wnt signaling pathway related gene sets from the scRNA-seq dataset, grouped by the FABP7 expression. ( C ) Relative luciferase assay examines effects on Wnt/β-catenin activation by over-expression (left) or silencing (right) of FABP7. Error bars represent the mean ± SD derived from three independent experiments. Statistical analyses were performed by two-sided Mann-Whitney test, *: p < 0.05. ( D ) Relative mRNA levels of Wnt/β-catenin downstream targets in FABP7 overexpressing (left) or silencing cells (right). Error bars represent the mean ± SD derived from three independent experiments. Statistical analyses were performed by two-sided Mann-Whitney test (*: p < 0.05). ( E ) Expression correlations between FABP7 and Wnt/β-catenin signaling targets in NSCLC cohort from TCGA dataset. ( F ) Representative images of immunohistochemistry consecutive slices staining (insets: high-magnification images) showing protein levels of FABP7 and Wnt/β-catenin signaling target genes in primary NSCLC tumors with low ( n = 10) or high ( n = 10) FABP7 expressions. Scale bar: 50 μm. Chi-square tests, *: p < 0.05. ( G ) Nucleo-plasma separation and western blotting assays show the changes of β-catenin expression at subcellular level by FABP7 ectopic expression in A549 cells. Representative images from three independent experiments are shown. ( H ) Representative IF images of three independent experiments (insets: high-magnification images) and quantitative results of alterations in β-catenin subcellular localization by FABP7 overexpression in A549 cells. Scale bar: 10 μm. Error bars represent the means ± SD derived from three independent experiments. Statistical analyses were performed by two-sided Mann-Whitney test (*: p < 0.05).

Article Snippet: Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively.

Techniques: Expressing, Luciferase, Activation Assay, Over Expression, Derivative Assay, MANN-WHITNEY, Immunohistochemistry, Staining, Western Blot

Journal: Cells

Article Title: Metastatic Tumor Cell-Specific FABP7 Promotes NSCLC Metastasis via Inhibiting β-Catenin Degradation

doi: 10.3390/cells11050805

Figure Lengend Snippet: FABP7 is important for the activation of Wnt/β-catenin signaling. ( A ) Tanswell assays were performed in indicated NSCLC cells. Representative images from three independent experiments (left) are shown. Migratory or invasive cells were quantified in five random fields. Scale bar: 50 μm. *: p < 0.05 vs. vector + negative control (NC), #: p < 0.05 vs. FABP7+NC. ( B ) Western blot analyses of EMT markers in indicated NSCLC cells. Representative images from three independent experiments are shown. ( C , D ) Results of relative luciferase assays ( C ) and representative IF images (insets: high-magnification images) from three independent experiments ( D ) in H1975 cells. *: p < 0.05 vs. vehicle+NC, #: p < 0.05 vs. Wnt3a+NC. Scale bar: 10 μm. ( E ) Tanswell assays were performed in indicated NSCLC cells. Representative images are from five random fields of transwell assay. Migratory or invasive cells were quantified in five random fields. Scale bar: 50 μm. *: p < 0.05 vs. vehicle+NC, #: p < 0.05 vs. Wnt3a+NC. ( F ) Western blot analyses on EMT markers in indicated NSCLC cells. Representative images are shown from three independent experiments. All error bars represent the mean ± SD derived from three independent experiments, and statistical analyses were performed by two-sided Mann-Whitney test.

Article Snippet: Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively.

Techniques: Activation Assay, Plasmid Preparation, Negative Control, Western Blot, Luciferase, Transwell Assay, Derivative Assay, MANN-WHITNEY

Journal: Cells

Article Title: Metastatic Tumor Cell-Specific FABP7 Promotes NSCLC Metastasis via Inhibiting β-Catenin Degradation

doi: 10.3390/cells11050805

Figure Lengend Snippet: FABP7 stabilizes β-catenin by inhibiting its ubiquitin-proteasomal degradation. ( A ) Western blotting analyses on β-catenin levels in indicated NSCLC cells stably expressing vector or FABP7. ( B ) Western blotting analyses (left) and corresponding quantitation (right) on β-catenin stability in 293FT cells with indicated treatments in the presence of CHX. Error bars represent the means ± SD derived from three independent experiments, and statistical analyses were performed by two-sided Mann-Whitney test (*: p < 0.05). ( C ) Alteration of β-catenin stability after indicated treatments in the presence of MG132 with or without FABP7 depletion by Western blotting analyses. ( D ) Co-immunoprecipitation (co-IP) assays in stable FABP7 knockdown A549 cells show changes of β-catenin polyubiquitination in the presence of MG132. ( E ) Interaction between FABP7 and purified β-catenin protein in A549 cells. ( F ) Interaction between β-catenin and destruction complex components upon FABP7 overexpression in A549 cells. ( G ) Co-IP assays show changes in β-catenin phosphorylation by FABP7 overexpression in A549 cells. Representative images are from three independent experiments.

Article Snippet: Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively.

Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Derivative Assay, MANN-WHITNEY, Immunoprecipitation, Co-Immunoprecipitation Assay, Purification, Over Expression