Invasion Assay of Lung Cancer Cells

For invasion assays, transiently transfected or stable knockdown of PPAT, PAICS and PKM2 in were used in A549 cells. BEAS-2B (benign transformed lung epithelial) cells were infected with PAICS-adenovirus. Seventy-two hours post-transfection and/or infection, cells were seeded onto BD BioCoat matrigel matrix (BD, CA) present in the insert of a 24-well culture plate. For A549, 10% serum containing RPMI medium was added to the lower chamber as a chemoattractant. For BEAS-2B, BEBM medium with supplementary growth factors was added as chemoattractant. After 36 h, the non-invading cells and matrigel matrix were gently removed with a cotton swab. Invasive cells located on the lower side of the chamber were stained with 0.2% crystal violet in methanol, air-dried and photographed. They were then enumerated microscopically using multiple representative areas. For colorimetric assays, the inserts were treated with 150 μl of 10% acetic acid and the absorbance measured at 560 nm [71 (link)]. Each experiment has been carried twice in triplicates per sample. Data shown is representative figure of one such experiment.