Journal: Advanced Science

Article Title: Detachment‐Induced FAK‐STAT3‐NNMT Inhibits CTCs Anoikis to Promote Breast Cancer Metastasis by Enhancing Fatty Acid Oxidation

doi: 10.1002/advs.202522837

Figure Lengend Snippet: Detachment‐induced NNMT is upregulated by the FAK‐STAT3 axis. (A) Representative image of the positive correlation between FAK and STAT3 in breast cancer and pan‐cancer according to the GEPIA 2 database analysis. (B) Representative results of STAT3, P‐STAT3, FAK, and P‐FAK proteins by WB in the detached BT‐549, MCF‐7, and MDA‐MB‐231 cells. (C,D) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m ) and P‐STAT3 inhibitor C188‐9 (10 µ m ) for 48 h by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (E,F) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m for 48 h) and P‐STAT3 activator (1 µ m for 24 h) by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (G,H) ChIP assay using P‐STAT3 antibodies in MDA‐MB‐231 cells cultured under suspension conditions was performed, followed by PCR and qPCR, which showed P‐STAT3 enrichment at the NNMT promoter vs. IgG control. ** p < 0.01. I: Dual‐luciferase reporter assays showed that co‐transfection with the full‐length NNMT promoter and a STAT3‐expressing plasmid significantly enhanced luciferase activity, whereas this enhancement was abolished when a truncated NNMT promoter was used. Data were presented as mean ± SEM. **** p < 0.0001.

Article Snippet: All plasmids used in this assay, including the firefly luciferase reporter vectors (e.g., pGL4‐basic containing the wild‐type or truncated NNMT promoter), the STAT3 expression plasmid, and the Renilla luciferase internal control plasmid (pRL‐TK), were custom‐synthesized by GeneChem Co., Ltd. (Shanghai, China).

Techniques: Cell Culture, Suspension, Control, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Activity Assay