Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: Expression, purification, and functional validation of recombinant FAT2 protein. (A) Agarose gel electrophoresis of the pET-30a-fat2 plasmid digested with NdeI and XhoI, confirming successful cloning. (B) SDS-PAGE analysis showing induced expression of recombinant FAT2 protein in E. coli BL21(DE3) (arrow indicates the target band). (C) Evaluation of the FAT2 purification process by Ni-NTA affinity chromatography. (D) SDS-PAGE analysis of the final purified FAT2 product. (E) Western blot analysis of purified FAT2 using an anti-His tag antibody, confirming protein identity. (F, G) CCK-8 assays showing that recombinant FAT2 protein at 1 μg/mL significantly inhibits the viability of BT-474 (F) and MDA-MB-453 (G) cells at 24 and 48 h, while lower concentrations (0.05–0.5 μg/mL) exert no significant cytotoxic effect. Data are presented as mean ± SD from at least five replicates. * P < 0.05, ** P < 0.01 vs. vehicle control.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Purification, Functional Assay, Biomarker Discovery, Recombinant, Agarose Gel Electrophoresis, Plasmid Preparation, Cloning, SDS Page, Affinity Chromatography, Western Blot, CCK-8 Assay, Control

Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: Recombinant FAT2 protein is internalized by HER2-positive breast cancer cells and suppresses migration and invasion of BT-474 cells. (A, B) Trypsin protection assay in BT-474 (A) and MDA-MB-453 (B) cells. Cells were incubated with His-FAT2 recombinant protein (0.5 μg/mL) and analyzed by Western blot using an anti-His antibody. Ctrl, untreated control; 37 °C, His-FAT2 incubation without trypsin; 37 °C + Tryp, His-FAT2 incubation followed by trypsin digestion at 37 °C; 4 °C + Tryp, His-FAT2 incubation followed by trypsin digestion at 4 °C. Trypsin-resistant bands indicate internalized protein. (C) Representative images of wound-healing assay showing the effect of recombinant FAT2 protein on BT-474 cell migration at 0 and 24 h. (D) Quantification of Transwell migration and Matrigel invasion assays demonstrating the inhibitory effects of FAT2 protein on BT-474 cell motility and invasiveness. Data are presented as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001 versus vehicle control.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Recombinant, Migration, Incubation, Western Blot, Control, Wound Healing Assay

Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: Recombinant FAT2 protein suppresses migration, invasion, and epithelial–mesenchymal transition (EMT) in breast cancer cells. (A) Transwell migration and invasion assays demonstrating that FAT2 treatment dose-dependently inhibits the migratory and invasive capacities of MDA-MB-453 cells. (B, C) Western blot analysis of EMT-related markers (E-cadherin, N-cadherin, Vimentin, and MMP9) in BT-474 (B) and MDA-MB-453 (C) cells treated with the indicated concentrations of FAT2 for 24 h. Data are presented as mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.001 vs. untreated control.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Recombinant, Migration, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: FAT2 positively regulates CLDN19 expression in breast cancer. (A) Gene Set Enrichment Analysis (GSEA) of FAT2-associated gene expression in the TCGA-BRCA dataset, showing significant enrichment of cell adhesion–related pathways. (B) Scatter plot showing the positive correlation between FAT2 and CLDN19 mRNA expression levels in breast cancer (Spearman’s r = 0.663, P < 0.001; TCGA data). (C, D) CLDN19 mRNA expression is significantly downregulated in breast cancer tissues compared with adjacent normal tissues (TCGA data). (E–G) Western blot (E, F) and qPCR (G) analyses showing dose-dependent upregulation of CLDN19 protein and mRNA levels in BT-474 and MDA-MB-453 cells treated with recombinant FAT2 protein (0, 0.25, 0.5 μg/mL) for 24 h. Data are presented as mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Gene Expression, Western Blot, Recombinant

Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: Knockdown of FAT2 promotes cell migration and downregulates CLDN19 expression. (A) Western blot validation of FAT2 knockdown efficiency in BT-474 and MDA-MB-453 cells transfected with FAT2-targeting siRNAs or negative control siRNA (siNC). (B) Transwell migration assay showing that FAT2 knockdown significantly promotes BT-474 cell migration. Quantification is shown in the right panel. (C, D) Western blot analysis demonstrating reduced CLDN19 protein expression following FAT2 knockdown in BT-474 (C) and MDA-MB-453 (D) cells. Data are representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. siNC group.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Migration, Expressing, Western Blot, Biomarker Discovery, Transfection, Negative Control, Transwell Migration Assay

Journal: Frontiers in Oncology

Article Title: FAT2 inhibits breast cancer cell migration, invasion, and epithelial–mesenchymal transition through transcriptional upregulation of CLDN19

doi: 10.3389/fonc.2026.1734983

Figure Lengend Snippet: CLDN19 mediates the anti-migratory and anti-EMT effects of FAT2 in breast cancer cells. (A) Western blot analysis confirming CLDN19 knockdown efficiency in BT-474 cells transfected with three independent siRNAs targeting CLDN19. (B) Western blot analysis of Vimentin expression in BT-474 cells 48 h after transfection with siCLDN19-1. (C, D) Transwell migration assay showing that concurrent CLDN19 knockdown partially rescues the inhibition of BT-474 cell migration induced by recombinant FAT2 protein (0.5 μg/mL). Representative images (C) and quantification (D) are shown. (E) Western blot analysis demonstrating that CLDN19 knockdown partially reverses the FAT2-induced downregulation of Vimentin. Data are representative of three independent experiments and presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. siNC group; # P < 0.05, ### P < 0.001 vs. FAT2 (0.5 μg/mL) + siCLDN19–1 group.

Article Snippet: Human breast cancer cell lines BT-474 (ER + ) and MDA-MB-453 (HER2 + ) were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Knockdown, Transfection, Expressing, Transwell Migration Assay, Inhibition, Migration, Recombinant