Transwell Invasion Assay of A549 Cells

Transwell invasion assay was performed using our modified protocol [57 (link),67 (link)]. Briefly: Nunc Cell Culture Inserts (transwell) with 8.0μm pore diameter (#141006) were covered with 50 µL 0.2% gelatin 1 h 37 °C. Next, gelatin was carefully removed. A549 or A549CisR cells were treated with or without 3 µM #19 for 24 h. Then, they were trypsinized, washed 2× with medium, and transferred (2 × 10⁵ cells/chamber) to upper chamber of Nunc Cell Culture Inserts in 0.1% BSA medium—supplemented with or without 3 µM #19. Full medium in lower chamber was used as chemoattractant. Next, medium and the gelatin from the top surface of the membrane were removed, invaded cells on the bottom surface of the membrane were washed 2× with PBS and then fixed for 5 min with CellFIX^TM. Cells were dyed at RT 15 min with Hoechst 33,342 (Molecular Probes/Life Technologies, Waltham, MA, USA). Finally, membranes were cut out from chambers and placed on microscope glass, and number of cells that migrate into the membrane was counted in 5 random spots.