Biocoat control insert no ecm

Transwell assays (Corning BioCoat Control Insert‐No ECM, 8 µm pore size) were performed according to the manufacturer's instructions. Briefly, A549 (1 × 10⁵) or SK‐Hep1 (1 × 10⁵) cells were collected after trypsinization and added into Transwell chamber of 6.5 mm diameter with serum‐free medium. And medium with 10% FBS was used in the outside of the chamber. 12 h later, the cells were fixed by methanol and stained with 0.4% crystal violet. Non‐migrated cells on the upper side of the chamber were erased by a cotton swab. Migrated cells were photographed by inverted microscope and relative cell migration was calculated by the area of migrated cells and normalized to the control group. For each experiment, the area of migrated cells in more than three random fields (magnification, 200×) on the underside of the chambers was counted, and three independent experiments were analyzed.

For RNA‐sequencing samples preparation, A549 (1 × 10⁶) cells were collected after trypsinization and added into Transwell chamber of 24 mm diameter (Corning BioCoat Control Insert‐No ECM, 8 µm pore size) with serum‐free medium. And medium with 20% FBS was used in the outside of the chamber. 18 h later, the membrane of chamber was immerged into trypsin and the cells adherent to the upper surface of the membrane and confined in the pore were collected separately. RNA of the collected cells was extracted with Trizol reagent (Life technologies).