GAPDH inhibition in A549 and H358 lung cancer cells

Human lung cancer cell lines A549 and H358 were obtained from (Biobank of Medical University of Graz) and cultured in RPMI 1640 (R0883; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco), 5 mM glutamine and 5 mM Pen Step, and sub-cultured regularly. For siRNA mediated knock-down of GAPDH, 150,000 A549 and H358 cells were seeded in triplicates of a six-well plate and transfected upon reaching ca. 60% confluency with silencerTM siRNA kit targeting GAPDH or non-target control (AM4605; Thermo), using RNAiMax (Thermo) and according to the manufacturer’s instructions for 6-well plate scale. For GAPDH inhibition, 150.000 A549 and H358 cells were seeded in six replicates per condition, then treated with 10 µM koningic acid (KA) or vehicle control (dmso). 48 h post transfection or 24 h post KA treatment, cells were harvested in 500 µl of harvesting solution (80% methanol in 50 mM ammonium acetate with 2.5 mM N-ethylmaleimide) and processed according to one-pot redox approach.