Evaluating AURKB and RSK Inhibitors on A549 Cells

A549 (2 × 10³) cells were seeded in 80 µL of DMEM supplemented with 10% FBS in a 96-well plate. After overnight incubation, 80 µL of media containing either 0.01% (v/v) DMSO or 1 µM Barasertib (AURKB inhibitor) or 5 µM BI-D1870 (RSK inhibitor) was added to the cells. In parallel, cells were also transfected with 25 ng of either Vo or YB-1:EBFP2 using Lipofectamine 3000 (LF3000, Invitrogen) as described above. Each treatment had six biological replicates. Twenty microliters of the lipoplexes were added to the cells. At the indicated time points the medium was removed and the plates were frozen at –80 °C. After all the time points had been collected the plates were thawed and the DNA content measured using a SYBR Green I-based fluorimetric assay as described previously [74 (link)]. Briefly, 100 µL of lysis buffer (10 mM Tris-HCl pH 8.0, 2.5 mM EDTA and 1% (v/v) Triton X-100) containing 1:4000 SYBR Green I (Invitrogen) was added to the wells and the plates were incubated overnight at 4 °C. The plates were mixed and the fluorescence signal for each well was measured for 1 s at an excitation of 485 nm and emission of 535 nm using a BioTek microplate reader (BioTek, Winooski, VT, USA). Growth curves were plotted as the fluorescence values at each time point. There were at least three biological replicates for the control and the treated samples.