Transwell migration assay and xenograft studies

A 24-well chemotaxis chamber (8-µm pore size; Corning) was used in this experiment. Briefly, 2×10⁴ cells of MDA-MB-231 and 4×10⁴ cells of HCC-1937 were loaded onto the upper well of the Transwell chamber with serum-free culture medium, while the medium containing 2.5% FBS for MDA-MB-231 and 10% FBS for HCC-1937 as an attractant added to the bottom chamber. After incubating plates for 24 h at 37°C, migration was assessed using the trypan-blue dye method.

Athymic nude (nu/nu) mice, female, 4–6 weeks of age, were purchased from Vital River and housed in a sterile room at the Experimental Animal Center of CMU University at 25°C and 40–70% humidity, with a 12-h light/dark cycle and free access to food and water. Lentivirus-transfected MDA-MB-231 cells were harvested. The concentration of cell suspensions was adjusted to 2×10⁶ cells/100 µl with PBS, and then injected into the caudal vein of nude mice. At least 5 mice were used for each group. Mice were monitored for symptoms related to tumor growth including an arched back, unsteady gait, and paralysis of legs and body weight loss each week. Eight weeks later, no mice died. The method of euthanasia was cervical dislocation (when the heart stopped completely, the mouse was determined as dead) after a total body weight loss of 20% or severe symptoms were observed. Lung tissues were fixed in 4% paraformaldehyde for later staining of pathological tissue sections. Hematoxylin and eosin (H&E) staining was used for verifying cancer tissues and confirming the pulmonary metastatic lesions under a microscope. IHC staining was used for checking the expression of FEN1. Anti-human FEN1 (working concentrations were 1:150) were purchased from ZSGB-Bio (TA802762). The mice were housed and maintained under standard NIH protocol.