Culturing A549 Lung Cancer Cells

A549 cells derived from human lung adenocarcinoma (RIKEN BRC through the National Bio-Resource Project of the MEXT, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS, Biological Industries, Israel) as described previously [41 (link)]. In two-dimensional (2D) and three-dimensional (3D) models, the cells were grown on flat-bottomed and PrimeSurface 96U plates (Sumitomo Bakelite, Tokyo, Japan), respectively. CLDN2 promoter construct and internal control pRL-TK vector were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan). The pTRE2 (mock) and CLDN2/pTRE2 mammalian expression vectors were transfected into spheroid cells using ScreenFect A (Fujifilm Wako Pure Chemical Industries).